阅读说明：本技术 两性离子共聚物涂料及其相关方法 (Zwitterionic copolymer coatings and methods relating thereto ) 是由 江绍毅 林晓杰 乔纳森·希梅尔法布 巴迪·D·拉特纳 于 2020-03-13 设计创作，主要内容包括：两性离子羧基甜菜碱共聚物及其在涂料中赋予表面,特别是血液接触医疗器械的表面防污和功能性的用途。(Zwitterionic carboxybetaine copolymers and their use in coatings to impart soil resistance and functionality to surfaces, particularly to surfaces of blood-contacting medical devices.)

1. A method of inhibiting or preventing leaching of a plasticizer from a substrate surface comprising:

(a) coating at least a portion of a surface of a substrate with a composition comprising a copolymer to provide a coated surface, the copolymer comprising a first repeating unit and a second repeating unit, wherein each of the first repeating units comprises a pendant zwitterionic group, and wherein each of the second repeating units comprises a pendant photoreactive group effective to crosslink the copolymer on the surface to provide the coated surface; and

(b) irradiating the coated surface with ultraviolet light effective to crosslink the copolymer on the surface, thereby providing a coated surface effective to inhibit or prevent leaching of plasticizer from the surface.

2. The method of claim 1, wherein the surface is a blood-contacting surface.

3. The method of claim 1, wherein the substrate is a polyurethane tube, a polysulfone dialysis membrane, or a hydrocarbon-based membrane container.

4. The method of claim 1, wherein coating the surface with the composition comprises dipping the surface into the composition.

5. The method of claim 1, wherein coating at least a portion of the surface comprises spraying, spin coating, brushing, or rolling the composition onto the surface.

6. The method of claim 1, wherein the surface is a hydrocarbon-based surface.

7. The method of claim 1, wherein the surface is a cellulosic, cellulose acetate, polyolefin, polyester, polycarbonate, Polyurethane (PU), Polysulfone (PSF), poly (ether sulfone) (PES), polyamide, polyacrylic, polyimide, aromatic polyester, Polyethylene (PE), polypropylene (PP), Polystyrene (PS), poly (ethylene terephthalate) (PET), polyvinyl chloride (PVC), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), or poly (methyl methacrylate) (PMMA) surface.

8. The method of claim 1, wherein the surface is a polyvinyl chloride surface or a polyurethane surface.

9. The method of claim 1, wherein the surface is a surface of a polyvinyl chloride pipe or a polyurethane pipe.

10. The method of claim 1, wherein the copolymer further comprises third repeating units, wherein each of the third repeating units comprises a pendant hydrophobic group effective to adsorb the copolymer to a plastic surface.

11. The method of claim 1, wherein the copolymer has formula (III):

wherein

R₁And R₂Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₃and R₄Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.10 to about 0.90 mol%;

a + b is 1.0; and

represents the copolymer end groups.

12. The method of claim 11, wherein R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, and X is NH and Y is NH.

13. The method of claim 11, wherein R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, X is O, and Y is O.

14. The method of claim 11, wherein n is 2 or 3.

15. The method of claim 11, wherein m is 1 or 2.

16. The method of claim 11, wherein a is about 0.70 to about 0.90 mol%.

17. The method of claim 11, wherein a is about 0.80 mol%.

18. The method of claim 11, wherein b is from about 0.10 to about 0.30 mol%.

19. The method of claim 11, wherein b is about 0.20 mol%.

20. The method of claim 11, wherein R₁Is hydrogen, R₂Is hydrogen, R₃And R₄Is methyl, X is NH, Y is NH, n is 3, and m is 1.

21. The method of claim 20, wherein a is about 0.80 mol%.

22. The method of claim 20, wherein b is about 0.20 mol%.

23. A copolymer for coating a surface, the copolymer comprising a first repeat unit and a second repeat unit, wherein each of the first repeat units comprises a pendant zwitterionic group, and wherein each of the second repeat units comprises a pendant photoreactive group effective to crosslink the copolymer.

24. The copolymer of claim 23, further comprising third repeat units, wherein each of the third repeat units comprises a hydrophobic group effective to adsorb the copolymer to a surface.

25. A copolymer for coating a surface, said copolymer comprising a first repeat unit, an optional second repeat unit, and an optional third repeat unit, with the proviso that said copolymer comprises at least a first repeat unit and an optional second repeat unit or at least a first repeat unit and an optional third repeat unit, wherein each of said first repeat units comprises a pendant zwitterionic group, wherein each of said second repeat units comprises a pendant photoreactive group effective to crosslink said copolymer, and wherein each of said third repeat units comprises a pendant hydrophobic group effective to adsorb said copolymer to a surface.

26. A copolymer for coating a surface, the copolymer being represented by formula (I):

*-(P₁)_a(P₂)_x(P₃)y-* (I)

wherein

P₁Are repeating units having pendant zwitterionic groups,

P₂are repeating units having pendant hydrophobic groups,

P₃having a repeating unit pendant from the photoreactive group,

a is from about 0.10 to about 0.90 mol%,

x is from 0 to about 0.95 mol%,

y is from 0 to about 0.95 mol%,

provided that x and y are not both 0, and

a + x + y is 1.0.

27. A copolymer of formula (II):

wherein

R₁And R₂Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₃and R₄Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

p is an integer of 0 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.10 to about 0.90 mol%;

a + b is 1.0; and

represents the copolymer end groups.

28. The copolymer of claim 27, wherein R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, and X is NH and Y is O.

29. The copolymer of claim 27, wherein R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, X is O, and Y is O.

30. The copolymer of any one of claims 27 to 29, wherein n is 2 or 3.

31. The copolymer of any one of claims 27 to 29, wherein m is 1 or 2.

32. The copolymer of any one of claims 27 to 31, wherein p is 3.

33. The copolymer of any one of claims 27 to 32, wherein a is about 0.20 to about 0.40 mol%.

34. The copolymer of any one of claims 27 to 32, wherein a is about 0.30 mol%.

35. The copolymer of any one of claims 27 to 32, wherein b is from about 0.60 to about 0.80 mol%.

36. The copolymer of any one of claims 27 to 32, wherein b is about 0.70 mol%.

37. The copolymer of claim 27, wherein R₁Is hydrogen, R₂Is methyl, R₃And R₄Is methyl, X is NH, Y is O, n is 3, m is 1, and p is 2.

38. The copolymer of claim 37, wherein a is about 0.30 mol%.

39. The copolymer of claim 27, wherein R₁Is methyl, R₂Is methyl, R₃And R₄Is methyl, X is O, Y is O, n is 2, m is 2, and p is 2.

40. The copolymer of claim 39, wherein a is about 0.30 mol%.

41. A copolymer of formula (III):

wherein

R₁And R₂Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₃and R₄Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.10 to about 0.90 mol%;

a + b is 1.0; and

represents the copolymer end groups.

42. The copolymer of claim 41, wherein R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, and X is NH and Y is NH.

43. The copolymer of claim 41, wherein R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, X is O, and Y is O.

44. The copolymer of any one of claims 41 to 43, wherein n is 2 or 3.

45. The copolymer of any one of claims 41 to 44, wherein m is 1 or 2.

46. The copolymer of any one of claims 41 to 45, wherein a is about 0.70 to about 0.90 mol%.

47. The copolymer of any one of claims 41 to 45, wherein a is about 0.80 mol%.

48. The copolymer of any one of claims 41 to 45, wherein b is from about 0.10 to about 0.30 mol%.

49. The copolymer of any one of claims 41 to 45, wherein b is about 0.20 mol%.

50. The copolymer of claim 41, wherein R₁Is hydrogen, R₂Is hydrogen, R₃And R₄Is methyl, X is NH, Y is NH, n is 3, and m is 1.

51. The copolymer of claim 50, wherein a is about 0.80 mol%.

52. The copolymer of claim 50, wherein b is about 0.20 mol%.

53. A copolymer of formula (IV):

wherein

R₁、R₂And R₃Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₄and R₅Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

z is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

p is an integer of 0 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.05 to about 0.95 mol%;

c is about 0.05 to about 0.95 mol%;

a + b + c is 1.0; and

represents the copolymer end groups.

54. The copolymer of claim 53, wherein R₁、R₂And R₃Independently is hydrogen or methyl, R₄And R₅Is methyl, and X is NH, Y is O and Z is NH.

55. The copolymer of claim 53 or 54, wherein n is 2 or 3.

56. The copolymer of any one of claims 53 to 55, wherein m is 1 or 2.

57. The copolymer of any one of claims 53 to 56, wherein p is 3.

58. The copolymer of any one of claims 53 to 57, wherein a is from about 0.70 to about 0.90 mol%.

59. The copolymer of any one of claims 53 to 57, wherein a is about 0.70 mol%.

60. The copolymer of any one of claims 53 to 59, wherein b is about 0.05 to about 0.25 mol%.

61. The copolymer of any one of claims 53 to 59, wherein b is about 0.20 mol%.

62. The copolymer of any one of claims 53 to 61, wherein c is about 0.05 to 0.20 mol%.

63. The copolymer of any one of claims 53 to 61, wherein c is about 0.10 mol%.

64. The copolymer of any one of claim 53, wherein R₁、R₂And R₃Is hydrogen, R₄And R₅Is methyl, X is NH, Y is O, Z is NH, n is 3, m is 1, and p is 3.

65. The copolymer of claim 64, wherein a is about 0.70 mol%, b is about 0.20 mol%, and c is about 0.10 mol%.

66. A coating composition comprising the copolymer of any one of claims 23 to 65.

67. A coating for a substrate surface comprising the copolymer of any one of claims 23 to 65 or derived from the copolymer of any one of claims 23 to 65.

68. A substrate having at least a portion or all of its surface coated with a composition comprising the copolymer of any one of claims 23 to 65 or derived from the copolymer of any one of claims 23 to 65.

69. The substrate of claim 68, wherein the surface is a hydrocarbon-based surface.

70. The substrate of claim 68, wherein the surface is a cellulosic, cellulose acetate, polyolefin, polyester, polycarbonate, Polyurethane (PU), Polysulfone (PSF), poly (ether sulfone) (PES), polyamide, polyacrylic acid, polyimide, aromatic polyester, Polyethylene (PE), polypropylene (PP), Polystyrene (PS), poly (ethylene terephthalate) (PET), polyvinyl chloride (PVC), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), or poly (methyl methacrylate) (PMMA) surface.

71. The substrate according to claim 68, wherein the substrate is a PVC tube, a polyurethane tube, a polysulfone dialysis membrane, or a hydrocarbon-based membrane container.

72. A medical device having at least a portion or all of its surface coated with a composition comprising the copolymer of any one of claims 23 to 65.

73. The apparatus of claim 72, wherein the surface is a hydrocarbon-based surface.

74. The device of claim 72, wherein the surface is a cellulosic, cellulose acetate, polyolefin, polyester, polycarbonate, Polyurethane (PU), Polysulfone (PSF), poly (ether sulfone) (PES), polyamide, polyacrylic, polyimide, aromatic polyester, Polyethylene (PE), polypropylene (PP), Polystyrene (PS), poly (ethylene terephthalate) (PET), polyvinyl chloride (PVC), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), or poly (methyl methacrylate) (PMMA) surface.

75. The device of claim 72, wherein the device is a plate, a disc, a tube, a tip, a catheter, an artificial blood vessel, an artificial heart, or an artificial lung.

76. The device of claim 72, wherein the device is an implantable/non-implantable medical device from class I, class II, or class III.

77. The device of claim 72, wherein the device is a hemodialyzer, a PVC tube, or a polyurethane tube.

78. A method for coating a surface of a substrate comprising contacting the surface with a composition comprising a surface of a substrate of the copolymer of any of claims 23 to 65.

79. A method for coating a surface of a substrate comprising:

(a) coating at least a portion of the surface of a substrate with a composition comprising the copolymer of any one of claims 23 to 26 or 41 to 65; and

(b) irradiating the surface of the substrate with light effective to crosslink the copolymer.

80. A method of antifouling a substrate surface comprising coating at least a portion of the surface with a composition comprising the copolymer of any of claims 23 to 65.

81. A method of antifouling a substrate surface comprising:

(a) coating at least a portion of the surface of a substrate with a composition comprising the copolymer of any one of claims 23 to 26 or 41 to 65; and

(b) irradiating the surface of the substrate with light effective to crosslink the copolymer.

82. A method of inhibiting adsorption of blood proteins to a substrate surface comprising coating at least a portion of the surface with a composition comprising the copolymer of any one of claims 23 to 65.

83. A method of inhibiting blood protein adsorption to a substrate surface comprising:

(a) coating at least a portion of the surface of a substrate with a composition comprising the copolymer of any one of claims 23 to 26 or 41 to 65; and

(b) irradiating the surface of the substrate with light effective to crosslink the copolymer.

84. A method of inhibiting or preventing leaching of a plasticizer from a substrate surface comprising:

(a) coating at least a portion of the surface of a substrate with a composition comprising the copolymer of any one of claims 23 to 65 to provide a coated surface.

85. A method of inhibiting or preventing leaching of a plasticizer from a substrate surface comprising:

(a) coating at least a portion of a surface of a substrate with a composition comprising the copolymer of any one of claims 23 to 26 or 41 to 65 to provide a coated surface; and

(b) irradiating the coated surface with light effective to crosslink the copolymer on the surface, thereby providing a coated surface effective to inhibit or prevent leaching of plasticizer from the surface.

86. The method of any one of claims 78 to 85, wherein contacting or coating the surface with the composition comprises immersing the surface in the composition.

87. The method of any one of claims 78 to 85, wherein contacting or coating the surface with the composition comprises spraying, spin coating, brushing, or rolling the composition onto the surface.

88. The method of any one of claims 78 to 85, wherein the surface is a hydrocarbon-based surface.

89. The method of any one of claims 78 to 85, wherein the surface is a cellulosic, cellulose acetate, polyolefin, polyester, polycarbonate, Polyurethane (PU), Polysulfone (PSF), poly (ether sulfone) (PES), polyamide, polyacrylic acid, polyimide, aromatic polyester, Polyethylene (PE), polypropylene (PP), Polystyrene (PS), poly (ethylene terephthalate) (PET), polyvinyl chloride (PVC), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), or poly (methyl methacrylate) (PMMA) surface.

90. The method of any one of claims 78 to 85, wherein the surface is a polyvinyl chloride surface.

Background

Biofouling of the surface of medical devices in contact with blood has been a major problem with thrombosis (thrombosis). Protein adsorption is the major event that occurs at the surface of biological materials in contact with biological media. Non-specific protein adsorption causes biofouling, which ultimately leads to serious adverse biological reactions such as platelet activation, thrombosis, complement activation, and inflammatory reactions. Therefore, antithrombotic blood contact devices strive to effectively avoid non-specific protein adsorption. This is achieved by an ultra-low contamination interface between the blood components and the surfaces in contact therewith. Synthetic polymeric biomaterials with hydrophilic and charge neutral properties hold promise due to their high biocompatibility. Traditional biomaterials such as poly (2-hydroxyethyl methacrylate) poly (HEMA) and poly (ethylene glycol) (PEG) have historically been used as gold standards for surface modification. However, poly (HEMA) has low water content and poor resistance to protein adsorption, while PEG can cause adverse reactions, including the appearance of anti-PEG antibodies and PEG-induced histological changes.

It is known that an indestructible hydration layer formed on the surface of a polymer chain can efficiently repel adsorption of biomolecules. Thus, hydration-induced antifouling capacity is a major feature of hydrophilic antifouling polymeric biomaterials. Zwitterionic compounds containing superhydrophilic zwitterionic groups, particularly Phosphorylcholine (PC), Carboxybetaine (CB), and Sulfobetaine (SB), have become common blood-inert biomaterials over the past two decades. The superhydrophilic and charge neutral zwitterionic groups with internal salt structures can form a layer of strongly bound water molecules that cannot be replaced by biologically active species, thereby inhibiting non-specific protein adsorption. Recently, CB-based polymers have shown excellent hydration-induced anti-fouling capabilities in a variety of applications, which makes them very attractive for use with blood-contacting surfaces. In addition, the CB group has the unique ability to covalently immobilize biomolecules (such as proteins, enzymes and oligonucleotides) on its carboxyl group.

Surface modification using zwitterionic polymers can be achieved by either "grafting from" or "grafting to" methods. To achieve non-invasive surface coating of large molded medical devices with various shapes and sizes, the "grafting to" polymer appears to be more robust by simply applying various polymers (e.g., homopolymers, random copolymers, and di-/tri-/multi-block copolymers) with different structures (architecures) to various surfaces via silane, catechol, and light/heat induced covalent bonding. Among them, the random amphiphilic zwitterionic copolymer is a simple and effective antifouling material which can effectively resist protein adsorption and platelet adhesion on the blood contact surface for a long period of time. Poly (MPC-co-n-Butyl Methacrylate (BMA)) having about 30 mol% hydrophilic units has been applied to many surfaces of medical devices and has excellent biocompatibility. However, comprehensive studies on the CB random copolymer are still lacking at present. In addition, their ability to surface functionalize the terminal carboxyl groups makes them an attractive surface modification material.

Despite the advances in the development of anti-fouling polymeric materials and their use in surface coatings, there remains a need for improved materials and surface coatings, particularly improved materials that can be readily coated on surfaces to advantageously provide an anti-fouling surface for blood-contacting medical device surfaces. The present invention seeks to meet this need and provide further related advantages.

Disclosure of Invention

The present invention provides zwitterionic copolymers and their use in coatings to impart soil resistance and functionality to surfaces, particularly surfaces of medical devices that contact blood. In some aspects, the present invention provides zwitterionic copolymers functionalized for surface immobilization thereof, coating compositions comprising the zwitterionic copolymers, surface coatings prepared from the zwitterionic copolymers, substrates having surfaces modified with the zwitterionic copolymers, and medical devices having surfaces modified with the zwitterionic copolymers.

In one aspect, the present invention provides a method of inhibiting or preventing leaching of a plasticizer from a substrate surface, comprising:

(a) coating at least a portion of the surface of the substrate with a composition comprising a copolymer to provide a coated surface, the copolymer comprising a first repeat unit and a second repeat unit, wherein each of the first repeat units comprises a pendant zwitterionic group, and wherein each of the second repeat units comprises a pendant photoreactive group effective to crosslink the copolymer on the surface to provide the coated surface; and

(b) the coated surface is irradiated with light effective to crosslink the copolymer on the surface, thereby providing a coated surface effective to inhibit or prevent leaching of the plasticizer from the surface.

In some embodiments, the copolymer further comprises third repeating units, wherein each of the third repeating units comprises a pendant hydrophobic group effective to adsorb the copolymer to a surface.

Surfaces that are advantageously treated by the above-described methods include hydrocarbon-based surfaces, such as blood-contacting surfaces. Representative surfaces include polyolefin, polyester, polycarbonate, Polyurethane (PU), Polysulfone (PSF), poly (ether sulfone) (PES), polyamide, polyacrylic, polyimide, aromatic polyester, Polyethylene (PE), polypropylene (PP), Polystyrene (PS)), poly (ethylene terephthalate) (PET), polyvinyl chloride (PVC), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), and poly (methyl methacrylate) (PMMA) surfaces. In some embodiments, the surface is a polyvinyl chloride surface or a polyurethane surface. In other embodiments, the surface is a cellulose or cellulose acetate surface.

In some embodiments, the surface is a surface of a polyvinyl chloride tube or a polyurethane tube.

In other embodiments, the surface is a blood contact surface, such as a dialysis membrane or a hydrocarbon-based membrane container.

In the above method, coating the surface with the composition comprises immersing the surface in the composition. In other embodiments, coating at least a portion of the surface comprises spraying, spin coating, brushing, or rolling the composition onto the surface.

In some embodiments, the method utilizes a copolymer of formula (III):

wherein

R₁And R₂Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₃and R₄Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.10 to about 0.90 mol%;

a + b is 1.0; and

represents the copolymer end groups.

In some of these embodiments, R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, and X is NH and Y is NH. In still other of these embodiments, R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, X is O, and Y is O. In some embodiments, n is 2 or 3. In some embodiments, m is 1 or 2. In some embodiments, a is from about 0.70 to about 0.90 mol%. In some of these embodiments, a is about 0.80 mol%. In some embodiments, b is from about 0.10 to about 0.30 mol%. In some of these embodiments, b is about 0.20 mol%.

In one embodiment, the method utilizes a polymer of formula (III), wherein R₁Is hydrogen, R₂Is hydrogen, R₃And R₄Is methyl, X is NH, Y is NH, n is 3, and m is 1. In some of these embodiments, a is about 0.80 mol%. In some of these embodiments, b is about 0.20 mol%.

In other embodiments, the method utilizes a polymer of formula (IV):

wherein

R₁、R₂And R₃Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₄and R₅Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

z is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

p is an integer of 0 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.05 to about 0.95 mol%;

c is about 0.05 to about 0.95 mol%;

a + b + c is 1.0; and

represents the copolymer end groups.

In another aspect, the present invention provides zwitterionic copolymers useful for coating surfaces to impart antifouling properties to the coated surfaces.

In one embodiment, the present invention provides a copolymer comprising a first repeat unit and a second repeat unit, wherein each of the first repeat units comprises a pendant zwitterionic group, and wherein each of the second repeat units comprises a pendant photoreactive group effective to crosslink the copolymer. In related embodiments, the copolymer further comprises third repeat units, wherein each of the third repeat units comprises a hydrophobic group effective to adsorb the copolymer to a surface.

In another embodiment, the present invention provides a copolymer comprising a first repeat unit, an optional second repeat unit, and an optional third repeat unit, provided that the copolymer comprises at least the first repeat unit and the optional second repeat unit or comprises at least the first repeat unit and the optional third repeat unit, wherein each of the first repeat units comprises a pendant zwitterionic group, wherein each of the second repeat units comprises a pendant photoreactive group effective to crosslink the copolymer, and wherein each of the third repeat units comprises a pendant hydrophobic group effective to adsorb the copolymer to a surface.

In a further embodiment, the present invention provides a copolymer represented by formula (I):

*-(P₁)_a(P₂)_x(P₃)y-*(I)

wherein

P₁To have a side-linkageA repeating unit of an ionic group, wherein,

P₂are repeating units having pendant hydrophobic groups,

P₃having a repeating unit pendant from the photoreactive group,

a is from about 0.10 to about 0.90 mol%,

x is from 0 to about 0.95 mol%,

y is from 0 to about 0.95 mol%,

provided that x and y are not both 0, and

a + x + y is 1.0.

In another embodiment, the present invention provides a zwitterionic/hydrophobic copolymer having the formula (II):

wherein

R₁And R₂Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₃and R₄Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

p is an integer of 0 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.10 to about 0.90 mol%;

a + b is 1.0; and

represents the copolymer end groups.

In another embodiment, the present invention provides a zwitterionic/hydrophobic copolymer having formula (III):

wherein

R₁And R₂Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₃and R₄Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.10 to about 0.90 mol%;

a + b is 1.0; and

represents the copolymer end groups.

In a further embodiment, the present invention provides a zwitterionic/hydrophobic copolymer having formula (IV):

wherein

R₁、R₂And R₃Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₄and R₅Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

z is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

p is an integer of 0 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.05 to about 0.95 mol%;

c is about 0.05 to about 0.95 mol%;

a + b + c is 1.0; and

represents the copolymer end groups.

In further aspects, the present invention provides coating compositions comprising zwitterionic copolymers and coated surfaces prepared using the zwitterionic copolymers and coating compositions. In some embodiments, the coating composition comprises a zwitterionic copolymer as described herein and optionally a carrier. In some embodiments, the coating comprises a zwitterionic copolymer as described herein or derived from a zwitterionic copolymer as described herein (e.g., a photocrosslinked zwitterionic copolymer).

In some embodiments, the present invention provides a substrate having at least a portion or all of its surface coated with the zwitterionic copolymer of the invention. Suitable surfaces include hydrocarbon-based surfaces and plastic surfaces. Representative surfaces include polyolefin, polyester, polycarbonate, Polyurethane (PU), Polysulfone (PSF), poly (ether sulfone) (PES), polyamide, polyacrylic, polyimide, aromatic polyester, Polyethylene (PE), polypropylene (PP), Polystyrene (PS), poly (ethylene terephthalate) (PET), polyvinyl chloride (PVC), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), and poly (methyl methacrylate) (PMMA) surfaces. In some embodiments, the surface is a polyvinyl chloride surface or a polyurethane surface. In other embodiments, the surface is a cellulose or cellulose acetate surface.

In some embodiments, the substrate is a PVC tube, a polyurethane tube, a polysulfone dialysis membrane, or a hydrocarbon-based membrane container.

In some embodiments, the substrate is a medical device having at least a portion or all of its surface coated with a zwitterionic copolymer as described herein.

In some embodiments, the device is an implantable/non-implantable medical device from class I, class II, or class III. In some embodiments, the device is a plate, a disc, a tube, a tip, a catheter, an artificial blood vessel, an artificial heart, or an artificial lung. In other embodiments, the device is a hemodialyzer, PVC tubing, or polyurethane tubing.

In another aspect of the invention, a method of using the zwitterionic copolymer is provided.

In one embodiment, the present invention provides a method for coating a surface of a substrate comprising contacting the surface with a composition comprising the zwitterionic copolymer of the invention as described herein.

In a related embodiment, the present invention provides a method for coating a surface of a substrate comprising:

(a) coating at least a portion of the surface of a substrate with a composition comprising a zwitterionic copolymer as described herein; and

(b) irradiating the surface of the substrate with light effective to crosslink the copolymer.

In another embodiment, the present invention provides a method of antifouling a substrate surface comprising coating at least a portion of the surface with a composition comprising a zwitterionic copolymer as described herein.

In a related embodiment, the present invention provides a method of antifouling a substrate surface comprising:

(a) coating at least a portion of the surface of a substrate with a composition comprising a zwitterionic copolymer as described herein; and

(b) irradiating the surface of the substrate with light effective to crosslink the copolymer.

In another embodiment, the present invention provides a method of inhibiting blood protein adsorption to a substrate surface comprising coating at least a portion of the surface with a composition comprising a zwitterionic copolymer as described herein.

In a related method, the present invention provides a method of inhibiting blood protein adsorption to a surface of a substrate, comprising:

(a) coating at least a portion of the surface of a substrate with a composition comprising a zwitterionic copolymer as described herein; and

(b) irradiating the surface of the substrate with light effective to crosslink the copolymer.

In another embodiment, the present invention provides a method of inhibiting or preventing the leaching of a plasticizer from a surface of a substrate comprising:

(a) coating at least a portion of a surface of a substrate with a composition comprising a zwitterionic copolymer as described herein to provide a coated surface.

In a related embodiment, the present invention provides a method of inhibiting or preventing leaching of a plasticizer from a surface of a substrate, comprising:

(a) coating at least a portion of a surface of a substrate with a composition comprising a photoreactive zwitterionic copolymer as described herein to provide a coated surface; and

(b) the coated surface is irradiated with light effective to crosslink the copolymer on the surface, thereby providing a coated surface effective to inhibit or prevent leaching of the plasticizer from the surface.

In some of the above methods, contacting or coating the surface with the composition comprises immersing the surface in the composition (e.g., zwitterionic copolymer). In other embodiments, contacting or coating a surface with the composition comprises spraying, rotating, brushing, or rolling the composition onto the surface.

Suitable surfaces include hydrocarbon-based surfaces and plastic surfaces. Representative surfaces include polyolefin, polyester, polycarbonate, Polyurethane (PU), Polysulfone (PSF), Polyethersulfone (PES), polyamide, polyacrylic acid, polyimide, aromatic polyester, Polyethylene (PE), polypropylene (PP), Polystyrene (PS), poly (ethylene terephthalate) (PET), polyvinyl chloride (PVC), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), and poly (methyl methacrylate) (PMMA) surfaces. In some embodiments, the surface is a polyvinyl chloride surface or a polyurethane surface. In other embodiments, the surface is a cellulose or cellulose acetate surface.

Drawings

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

FIG. 1 is a schematic illustration of the preparation of ultra-low contamination and functionalizable Carboxybetaine (CB) polymer surfaces according to the present invention.

FIGS. 2A and 2B show the chemical structures of representative Carboxybetaine (CB) random copolymers of the present invention: poly (CB1-co-BMA) (PCB1) (fig. 1A) and poly (CB2-co-BMA) (PCB2) (fig. 1B).Numbers 1 and 2 after CB represent the number of carbons between the carboxyl group and the quaternary ammonium cation. The composition of each unit in the copolymer is determined by¹Integral calculation of characteristic protons in H-NMR spectrum, with CB1 unit of 3.82ppm (-CH)₂-, 2H), CB2 unit was 2.42ppm (-CH)₂-, 2H), BMA unit of 1.45 to 1.63ppm (-CH)₂-, 4H). The carboxyl groups in PCB2 may be activated by EDC/NHS chemistry and covalently bonded to amino groups of, for example, proteins, enzymes, and aptamers/oligonucleotides. CB1 is carboxyl betaine acrylamide, 1-carboxyl-N, N-dimethyl-N- (3' -acrylamide propyl) ethylammonium inner salt (ethaneaminium inner salt); CB2 is carboxyl betaine methacrylate, 2-carboxyl-N, N-dimethyl-N- (2' -methacryloyloxyethyl) ethylammonium inner salt; BMA is n-butyl methacrylate.

FIG. 3 is a table summarizing the properties of representative carboxybetaine random copolymers of the invention.

FIG. 4 compares the relative adsorption of fibrinogen (1.0mg/mL, 1XPBS, pH7.4) on PP surfaces coated with representative carboxybetaine random copolymers of the invention, PCB1-37 and PCB2-37, at a concentration of 0.5 wt%.

Fig. 5A and 5B compare air contact angles measured in distilled water for polypropylene (PP) substrates modified with different concentrations of different CB random copolymers (5A) and PCB1-37 (5B). The numerical designation refers to the molar ratio of the two units in the copolymer (i.e., 28 in PCB1-28 indicates a molar ratio of CB/BMA of 2/8 in the copolymer). Single asterisk in 5A indicates statistically significant difference (p <0.05, n ═ 5) compared to PCB 1-37; the single asterisk in 5B indicates a statistically significant difference compared to 0.50 wt% (p <0.05, n ═ 5).

FIGS. 6A and 6B compare the relative adsorption of fibrinogen (1.0mg/mL, 1xPBS, pH7.4) on PP surfaces coated with different concentrations of different CB random copolymer (6A) and PCB1-37 (6B). The numerical designation refers to the molar ratio of the two units in the copolymer (i.e., 28 in PCB1-28 indicates a molar ratio of CB/BMA of 2/8 in the copolymer). Single asterisk in 5A indicates statistically significant difference (p <0.05, n ═ 5) compared to PCB 1-37; the single asterisk in 5B indicates a statistically significant difference compared to 0.50 wt% (p <0.05, n ═ 5). The relative absorbance at 492nm represents the relative adsorption amount of fibrinogen.

Fig. 7A to 7D are performed on 96-well plates having three surfaces: non-specific protein adsorption against (a) human plasma fibrinogen (Fg) (7A), Human Serum Albumin (HSA) (7B), human blood gamma-globulin (7C) and human serum (7D) was compared on uncoated polystyrene surfaces, PCB1-37 coated polystyrene surfaces and commercially available plates with ultra-low attachment surfaces (Corning Costar corp., Corning, NY, USA). A Corning ultra-low surface coating is a hydrophilic, neutral-charged coating covalently bonded to a polystyrene surface. Both 10% and 100% of single blood proteins and human serum were used to evaluate their anti-fouling ability. Uncoated surfaces and commercial products with ultra-low adhesion surfaces were used as controls. The concentrations of 100% Fg, HSA and gamma-globulin in blood were 3.0, 45 and 16mg/mL, respectively. Normal human serum (pooled mixed sex) was used as received without dilution. Single asterisk indicates statistically significant differences (p <0.01, n ═ 5) compared to uncoated or commercially available ultra-low adhesion surfaces; the double asterisk indicates a statistically significant difference (p <0.01, n ═ 5) compared to the uncoated surface.

FIG. 8 is a surface comparison graph illustrating the adhesion and migration behavior of NIH3T3 mouse embryonic fibroblasts on CB polymer (PCB1-37) coated TCPS surfaces (left) and pristine TCPS surfaces (right).

Figure 9 compares surface functionalization and specific antibody-antigen interactions on 96-well plates coated with CB random copolymer PCB 2-37. Both Fg and HSA were conjugated to the surface of CB polymer activated by EDC/NHS chemistry, respectively. anti-Fg conjugated with HRP was used to assess specific binding to further validate the feasibility of using 96-well plates for diagnosis. The absorbance at 492nm indirectly represents the amount of anti-Fg conjugated on the surface. The unactivated surface and the surface conjugated to HSA were used as controls. Single asterisk indicates statistically significant difference (p <0.001, n ═ 5).

Figure 10A is a schematic representation of a zwitterionic Carboxybetaine (CB) copolymer coated commercially available medical grade blood contact PVC tube.

FIG. 10B shows the chemical structure of a representative photoreactive Carboxybetaine (CB) polymer useful in the methods of the present invention.

Figure 11A shows UV light transmission through commercial medical grade PVC tubing.

Figure 11B shows the through light induced degradation of photosensitive (benzophenone) groups on PCB copolymers.

FIG. 12A shows a commercially available flat PVC pipe.

Fig. 12B compares the water contact angle on uncoated PVC film and PCB coated PVC film.

Fig. 13A to 13D compare X-ray photoelectron spectroscopy (XPS) measurement (surveyy) spectra of four of the following: representative PCB copolymer (13A), uncoated commercial PVC pipe (13B), PCB coated commercial PVC pipe stored for 1 week under dry conditions (13C), and PCB coated commercial PVC pipe stored for 3 weeks under dry conditions (13D).

Figure 14 compares the relative fouling of 100% human serum on PCB coated and uncoated medical grade PVC tubing.

Fig. 15A to 15C show the chemical structures of the following polymers: poly (CBAA-co-BPAA) (PCB) (15A); poly (CBAA-co-BPAA-co-NB acrylamide) (PCB-NB) (15B); and poly (PEGMA-co-BPAA) (PPB) (15C). CBAA: carboxybetaine acrylamide. BPAA: n- (4-benzoylphenyl) acrylamide. NB acrylamide: nile blue acrylamide. PEGMA: poly (ethylene glycol) methacrylate, average Mn 360.

Fig. 16A to 16H relate to light-induced crosslinking of PCB polymers on the inner tube surface: transmission light induced crosslinking (16A) of the PCB polymer on the inner tube surface; UV light transmission through commercial medical grade PVC tubing (16B); the photosensitivity of the PCB copolymer on the inner tube surface to transmitted light (16C); water contact angle of pure DI water on uncoated and PCB coated PVC surfaces (16D); x-ray photoelectron spectroscopy (XPS) measured spectra for the PCB copolymer (16E), uncoated commercial PVC pipe (16F), PCB coated commercial PVC pipe stored for 1 week under dry conditions (16G), and PCB coated commercial PVC pipe stored for 3 weeks under dry conditions (16H). The Binding Energy (BE) was corrected using the peak of C1s at 285eV as a reference.

Fig. 17A to 17F show antifouling ability evaluations: adsorption behavior of 100% human serum on PVC surfaces and PCB polymer coated PVC surfaces under dynamic conditions of four different flow rates (10, 40, 100 and 200 μ L/min) was tested by Surface Plasmon Resonance (SPR) biosensors (17A-17D). Different flow rates will produce different shear forces at the interface between the serum and the tube. Non-coating: SPR chips coated with PVC. PCB coated: SPR chip coated with a PVC first layer and a PCB polymer second layer. The thickness of the PVC and PCB were measured under dry conditions and were 18. + -. 0.8nm and 21. + -. 1.2nm, respectively. Figure 17F shows the adsorption of 100% human serum under static conditions tested using the Micro BCA protein assay kit. Non-coating: commercially available PVC pipe. PCB coated: the PCB-coated PVC tubes were soaked in PBS (1X, pH7.4) at 37 ℃ for 1 week or 3 weeks. Results are expressed as mean ± SD (n ═ 3;. p <0.001, and. p > 0.05).

Fig. 18A to 18C show cytotoxicity evaluation of PCB polymers. Phase-contrast microscopy images of NIH3T3 mouse embryonic fibroblasts eluted from PCB-coated tubes, uncoated tubes, latex and normal cell culture medium (18A) and cell culture medium containing different concentrations of PCB polymer (18B) after 48 hours of incubation. Scale bar: 50 μm. The cytotoxicity of PCB polymers was examined according to ISO10993-5 guidelines. Release of cytosolic enzyme Lactate Dehydrogenase (LDH) was achieved by contacting cells with different concentrations of PCB polymer at 37 ℃ in 5% CO₂And incubated for 1.0 hour (18C). Negative Control (NC): cells were cultured in normal serum-free cell culture medium. Positive Control (PC): cells were cultured with 0.2 vol% Tween 20 in serum free cell culture medium. All media were supplemented with phenol red and 1 × penicillin/streptomycin. The results are expressed as mean ± SD (n ═ 5;. p)<0.001, and>0.05)。

fig. 19A to 19D show the effect of PCB polymer on platelet quality. Activation levels were assessed by flow cytometry via p-selectin% (19A). Functionality was assessed by Von Willebrand Factor (VWF) binding affinity (19B). Viability was assessed by flow cytometry via annexin V% (19C). The Morphological Score (MS) was used as a simple indicator of platelet health and was defined as 4 × (dish%) +2 × (sphere%) + (branch%) (19D). PCB coated: PCB coated PVC surface. Uncoated: uncoated PVC surface. Results are expressed as mean ± SD (n ═ 5;. p <0.001, and. p > 0.05).

Figure 20 compares the migration of plasticizer in photoreactive PCB polymer coated plasticized PVC and uncoated plasticized PVC tubes. The absorbance at 275nm is directly proportional to the concentration of leached plasticizer. Results are expressed as mean ± SD (n ═ 3;. p <0.001, and. p < 0.05).

FIG. 21 compares the activation of different PVC tubes by the complement system: PCB coated, uncoated and PPB coated. The absorbance at 450nm is directly proportional to the concentration of the terminal complement complex (sC5 b-9). Results are expressed as mean ± SD (n ═ 3;. p <0.001, and. p > 0.05).

Fig. 22A is a schematic representation of surface modification of a representative commercially available platelet bag with a Carboxybetaine (CB) copolymer.

FIG. 22B shows the chemical structure of a representative zwitterionic Carboxybetaine (CB) copolymer of the present invention.

Fig. 22C shows photosensitivity of representative photoreactive CB copolymers.

Fig. 23A to 23F show representative surface modifications according to the present invention: (ii) osmotic light-induced degradation of UV light transmission and photosensitive (benzophenone) groups through commercially available platelet bags (23A); surface morphology of the platelet bags before and after coating (23B); x-ray photoelectron spectroscopy (XPS) measurement spectra, high resolution spectra of C1s, O1 s, and N1s, and atomic compositions of five different samples: (1) PCB copolymer, (2) commercially available platelet bags, (3) ethanol rinsed commercially available platelet bags, (4) PCB coated commercially available platelet bags, and (5) PCB coated commercially available platelet bags soaked in buffer (23C to 23E, respectively); and the static air contact angle in distilled water and the static water contact angle under dry conditions (23F).

FIGS. 24A to 24F show the antifouling capability assessment of representative zwitterionic Carboxybetaine (CB) copolymers of the present invention: adsorption of human blood proteins under dynamic conditions as tested by Surface Plasmon Resonance (SPR) (24A); adsorption of human blood proteins under static conditions tested using the Micro BCA protein assay kit (24B); adhesion behavior (24C) of mammalian cells (NIH3T 3); adhesion behavior of fresh human platelets (24D); adhesion and biofilm formation by gram-positive strains of Staphylococcus epidermidis (24E) and Pseudomonas aeruginosa (24F).

Fig. 25A through 25H provide an assessment of platelets stored according to a representative method of the invention: expression level of annexin V on preserved platelets (25A); expression level of p-selectin (CD62) on preserved platelets (25B); morphology score (25C) of preserved platelets; von Willebrand Factor (VWF) binding affinity (25D); time to reach 100nN threshold during embolism test (25E); and pH (25F), glucose level (25G) and lactate level (25H) in platelet preservation fluid.

Detailed Description

The present invention provides zwitterionic copolymers and their use in coatings to impart soil resistance and functionality to surfaces, particularly surfaces of medical devices that contact blood. The present invention provides zwitterionic copolymers functionalized for surface immobilization thereof, coating compositions comprising the zwitterionic copolymers, surface coatings prepared from the zwitterionic copolymers, substrates having surfaces modified with the zwitterionic copolymers, and medical devices having surfaces modified with the zwitterionic copolymers.

Zwitterionic copolymers

In one aspect, the present invention provides zwitterionic copolymers that are functionalized to immobilize them on a surface.

In some embodiments, the present invention provides a copolymer for coating a surface, the copolymer comprising a first repeat unit and a second repeat unit, wherein each of the first repeat units comprises a pendant zwitterionic group, and wherein each of the second repeat units comprises a pendant photoreactive group effective to crosslink the copolymer to a plastic surface. In some of these embodiments, the copolymer further comprises third repeat units, wherein each of the third repeat units comprises a hydrophobic group effective to adsorb the copolymer to a plastic surface.

In some embodiments, the present invention provides a copolymer useful for coating a surface, the copolymer comprising a first repeat unit, an optional second repeat unit, and an optional third repeat unit, with the proviso that the copolymer comprises at least the first repeat unit and the optional second repeat unit or at least the first repeat unit and the optional third repeat unit, wherein each of the first repeat units comprises a pendant zwitterionic group, wherein each of the second repeat units comprises a pendant photoreactive group effective to crosslink the copolymer to the surface, and wherein each of the third repeat units comprises a pendant hydrophobic group effective to adsorb the copolymer to the surface. In these embodiments, the copolymer is represented by formula (I):

*-(P₁)_a(P₂)_x(P₃)y-* (I)

wherein

P₁Are repeating units having pendant zwitterionic groups,

P₂are repeating units having pendant hydrophobic groups,

P₃having a repeating unit pendant from the photoreactive group,

a is from about 0.10 to about 0.90 mol%,

x is from 0 to about 0.95 mol%,

y is from 0 to about 0.95 mol%,

provided that x and y are not both 0, and

a + x + y is 1.0.

The copolymers of the present invention comprise repeat units derived from polymerizable monomers and comonomers. In the copolymer represented by the formula (I), the repeating unit P₁、P₂And P₃Together forming the backbone (backbone) of the copolymer. Each repeating unit includes a zwitterionic group, a hydrophobic group, or a photoreactive group. In some embodiments, each of the zwitterionic group, the hydrophobic group, and the photoreactive group is a pendant group (i.e., these groups pendant from the copolymer backbone). In some of these embodiments, the copolymer having pendant groups is composed of a copolymer having pendant groupsAnd preparing a polymerizable monomer and a comonomer for grafting groups.

The nature of the copolymer backbone is not critical so long as the backbone does not adversely affect the overall properties of the copolymer, the properties of the zwitterionic, hydrophobic and photoreactive groups of the copolymer. Suitable copolymer backbones include polyesters, polycarbonates, polyurethanes, polyureas, polysulfides, polysulfones, polyimides, polyepoxides, aromatic polyesters, celluloses, fluoropolymers, polyacrylic acids, polyamides, polyanhydrides, polyethers, vinyl polymers, phenolic resins, elastomers, and other addition polymers. Representative copolymer backbones include those described in detail herein.

As used herein, the term "zwitterionic group" refers to a group that includes at least two functional groups, one of which has a positive charge and one of which has a negative charge, and the net charge of the group is zero. Representative zwitterionic groups include Carboxybetaine (CB) groups, Sulfobetaine (SB) groups, Phosphobetaine (PHB) groups, and Phosphocholine (PC) groups. The zwitterionic groups impart low fouling and functionalizable properties to surfaces treated or coated with the copolymer.

The term "hydrophobic group" refers to a group that is hydrocarbon and non-polar in nature. The hydrophobic groups serve to facilitate binding of the copolymer to a surface treated with the copolymer (e.g., via hydrophobic-hydrophobic interactions). The nature of the hydrophobic groups of the copolymer is not critical so long as the groups achieve binding of the copolymer to the surface sufficient to meet the intended use of treating or coating the surface and do not adversely affect the performance of the zwitterionic or photoreactive groups of the copolymer. Suitable hydrophobic groups include hydrocarbon-containing groups such as alkyl and alkylene groups having two or more, three or more, or four or more carbons (e.g., ethyl, ethylene, propyl, propylene, butyl, butylene groups). Representative hydrophobic groups include C₃-C₂₀Alkyl (e.g., n-butyl), a benzene ring-containing group (e.g., phenyl), a fluorinated alkyl (e.g., trifluoromethyl), and a fluorinated aryl group.

The term "photoreactive group" refers to a group that becomes reactive to crosslinking upon irradiation with ultraviolet light. The pendant photoreactive groups (i.e., crosslinking groups) serve to facilitate bonding of the copolymer to a surface treated with the copolymer. The nature of the photoreactive groups of the copolymer is not critical so long as the groups effect crosslinking of the copolymer on the surface sufficient to stabilize the copolymer on the treated or coated surface and do not adversely affect the performance of the zwitterionic or hydrophobic groups of the copolymer. Suitable photoreactive groups include groups that effect crosslinking upon irradiation with light of an appropriate wavelength (e.g., ultraviolet, 250-370 nm). Suitable photoreactive groups are derived from aromatic ketone, azide, diazirine (diazodiazirine) groups. Representative photoreactive groups include benzophenone, acetophenone, phenyl azide, aryl azide (e.g., phenyl azide), azido-methyl-coumarin, anthraquinone, and psoralen derivatives.

The photoreactive groups of the zwitterionic polymers described herein effectively crosslink the copolymer. The photoreactive group is also effective to crosslink the zwitterionic copolymer to a surface that has been coated with the copolymer, wherein the surface includes C-H that is reactive with the photoreactive group. Representative surfaces to which the zwitterionic copolymers can be crosslinked include C-H containing surfaces such as polyethylene, polystyrene, polyethylene terephthalate, polyvinyl chloride, and cyclic polyolefin surfaces. The photoreactive zwitterionic copolymers described herein are not crosslinked with metal, metal alloy or ceramic surfaces that do not contain C-H reactive groups. The use of photoreactive zwitterionic copolymers as described herein provides a surface with a durable network polymer film.

In other embodiments, the zwitterionic copolymer includes a crosslinking group that is activated by light and heat. In a further embodiment, the copolymer comprises crosslinking groups activated by heat.

The copolymers of the present invention and those used in the process of the present invention include random copolymers (e.g., copolymers of formulae (I), (II), (III), and (IV)) prepared by copolymerization of comonomers (e.g., copolymerization of polymerizable monomers having zwitterionic groups, polymerizable monomers having hydrophobic groups, and polymerizable monomers having photoreactive groups). In the copolymer formulas described and shown herein, a repeating unit comprising a zwitterionic group, a repeating unit comprising a hydrophobic group, and a repeating unit comprising a photoreactive group are described. See, for example, formulas (I), (II), (III) and (IV). These descriptions are not intended to limit the nature of the copolymer; the copolymers described may be random copolymers having a specified number of repeat units (e.g., for formula (I), "a" represents a repeat unit comprising a zwitterionic group, "x" represents a repeat unit comprising a hydrophobic group, and "y" represents a repeat unit comprising a photoreactive group).

As noted above, the copolymers as described herein may include additional repeat units, so long as the additional repeat units do not interfere with or adversely affect the properties of the intended use of the copolymer. These copolymers of the present invention "comprise" repeating units comprising, for example, zwitterionic groups, repeating units comprising hydrophobic groups and repeating units comprising photoreactive groups, as well as other repeating units.

In some embodiments, the copolymers of the present invention include only repeating units that include, for example, zwitterionic groups, repeating units that include hydrophobic groups, and repeating units that include photoreactive groups. These copolymers of the present invention "consist of" repeating units comprising zwitterionic groups, repeating units comprising hydrophobic groups, and repeating units comprising photoreactive groups, and do not comprise other repeating units.

The zwitterionic copolymers of the present invention include up to about 90 mole% zwitterionic groups. For the zwitterionic copolymers of the present invention that include hydrophobic groups, the copolymer includes up to about 70 mol% hydrophobic groups. For the zwitterionic copolymers of the present invention that include photoreactive groups, the copolymer includes up to about 30 mole% photoreactive groups.

The zwitterionic copolymers of the present invention have a weight average molecular weight of from about 1,000 to about 2,000,000.

Zwitterionic/hydrophobic copolymers

In one embodiment, the present invention provides copolymers having zwitterionic groups and hydrophobic groups, each of which is pendant from the backbone of the copolymer (i.e., zwitterionic/hydrophobic copolymers). The pendant zwitterionic groups impart low fouling and functionalizable properties to surfaces treated or coated with the copolymer. The pendant hydrophobic groups serve to facilitate the binding of the copolymer to a surface treated with the copolymer (e.g., via hydrophobic-hydrophobic interactions). The relative amounts of the zwitterionic groups and hydrophobic groups pendant from the backbone of the copolymer can be adjusted via copolymer synthesis to achieve the desired degree of low contamination and functionalization and binding of the copolymer to the surface, each of which can be tailored to the nature (e.g., composition) of the surface to be treated or coated.

In some aspects, the present invention provides a simple and effective method of modifying hydrophobic materials using amphiphilic zwitterionic (e.g., carboxybetaine, CB) random copolymers via dip coating techniques. By adjusting the composition of hydrophilic units and hydrophobic units in the series of amphiphilic copolymers, the influence of the polymeric amphipathy on the antifouling capacity is explored, and the optimal composition of the surface coating is determined. 100% human serum adsorption was measured on CB random copolymer coated surfaces. The results were compared to those of a commercial 96-well plate with an "ultra-low attachment surface". In addition, the CB copolymer is subsequently surface functionalized with anti-fibrinogen through covalent bonds between carboxyl groups within the CB and amino groups within the antigen. Therefore, such CB polymer materials and modification techniques are expected to be useful in a wide range of applications including antifouling medical instruments and medical diagnostics.

In one embodiment, the present invention provides a copolymer of formula (II):

wherein

R₁And R₂Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₃and R₄Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

p is an integer of 0 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.10 to about 0.90 mol%;

a + b is 1.0; and

represents the copolymer end groups.

In some of these embodiments, R₁And R₂Independently selected from the group consisting of hydrogen and methyl.

In some embodiments, R₃And R₄Independently selected from the group consisting of hydrogen and C1-C3 alkyl.

In some embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, n is 2 or 3.

In some embodiments, m is 1, 2, 3, 4, 5, or 6. In some embodiments, m is 1 or 2.

In some embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 3.

In some embodiments, a is from about 0.20 to about 0.40 mol%. In some embodiments, a is about 0.30 mol%.

In some embodiments, b is from about 0.60 to about 0.80 mol%. In some embodiments, b is about 0.70 mol%.

In some embodiments, R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, and X is NH and Y is O.

In other embodiments, R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, X is O, and Y is O.

In further embodiments, R₁Is hydrogen, R₂Is methyl, R₃And R₄Is methyl, X is NH, Y is ON is 3, m is 1 and p is 2. In some of these embodiments, a is about 0.30 mol%.

In other embodiments, R₁Is methyl, R₂Is methyl, R₃And R₄Is methyl, X is O, Y is O, n is 2, m is 2, and p is 2. In some of these embodiments, a is about 0.30 mol%.

The following is a description of the preparation, characterization and use of representative zwitterionic/hydrophobic copolymers of the invention and their use in the compositions and methods of the invention.

The present invention provides an efficient surface modification method to render hydrophobic surfaces superhydrophilic using ultra-low contamination/functionalizable Carboxybetaine (CB) copolymers via dip coating techniques. A series of CB random copolymers with different amphiphilicities were synthesized and coated on hydrophobic polypropylene (PP) and Polystyrene (PS) surfaces. Each coating was screened for anti-fouling ability by enzyme-linked immunosorbent assay (ELISA) and further evaluated comprehensively for 100% human serum by Micro BCA protein assay kit. Random copolymers containing about 30 mol% of CB units exhibit superhydrophilicity with the highest air contact angle in DI water greater than 165 °, with the best anti-fouling ability against 100% human serum. The surface of the 96-well plate coated with the optimized CB random copolymer showed significantly better antifouling capacity than the commercial 96-well plate with ultra-low attachment surface. Adhesion of mouse embryonic fibroblasts (NIH3T3) was completely inhibited on the surface coated with CB random copolymer. Furthermore, the optimal antifouling CB copolymer surface was functionalized with antigen via covalent bonds, verifying its specific interaction with its antibodies. Thus, such CB random copolymers are capable of imparting ultra-low contamination and functionalizability to the hydrophobic surface of the blood contact device.

Representative polycarboxybetaines, their properties, their use in preparing antifouling surfaces, and the properties of coated surfaces are described below.

Synthesis of random copolymer. Polyolefins have been widely manufactured for biomedical applications. However, these hydrophobic polymeric hydrocarbon-based materials can cause non-specific protein adsorption, platelet activation, blood clotting, thrombosis, and other problems associated with biological contamination. For practical applications, it is highly desirable to use the simplest method to achieve the intended goal. Free radical polymerization is one of the most common and useful methods for polymerizing polymers from small-scale laboratory testing to large-scale industrial applications, particularly for the polymerization of vinyl monomers. Amphiphilic random copolymers were synthesized by conventional free radical polymerization methods using AIBN as a thermal free radical initiator. The viscosity of these reaction solutions gradually increased as polymerization proceeded at 65 ℃ indicating the conversion of monomer to copolymer. From¹The integral value of the characteristic peak in the H-NMR spectrum gives the unit fraction (fraction) of each monomer: CB1 unit 3.82ppm (-CH)₂-, 2H), CB2 unit was 2.42ppm (-CH)₂-, 2H), BMA unit of 1.45 to 1.63ppm (-CH)₂-, 4H). The chemical structures and synthetic details of these polymers are shown in fig. 2A and 2B and table 1 (fig. 3), respectively. The hydrophilic CB units and hydrophobic BMA units in the polymer chain are randomly distributed with an overall composition approximately equal to that of the monomer feed solution. The solubility of the CB random copolymer in aqueous solution depends mainly on the CB unit composition. Copolymers containing more than 30 mol% of CB units are readily soluble in aqueous solutions, while less than 30 mol% will become insoluble in water (table 1). Therefore, water-insoluble CB copolymers containing 30 mol% of CB units are the best coating materials among these copolymers. These results are matched with other random copolymers containing phosphorylcholine groups, which have been widely used for surface modification of biomedical devices.

Air contact angle and protein adsorption coating screening. Amphiphilic CB copolymers with different hydrophobic/hydrophilic compositions can impart different antifouling capabilities. Hydrophilic (CB) units can promote ion-induced hydration, which can strongly bind water molecules to improve antifouling capacity. However, strong hydration forces may cause the coating material to detach from the surfaceAnd (5) separating. Hydrophobic (BMA) units will strongly bind to hydrophobic substrate surfaces to stabilize CB units on the surface in aqueous solution and improve coating durability. Therefore, it is necessary to prepare these coating random polymers in the proper CB/BMA molar ratio to maximize antifouling and surface binding properties. The results of air contact angles in FIGS. 5A and 5B show that the copolymer containing about 30 mol% of CB units exhibits super hydrophilicity, and the air contact angle is as high as 165 deg.. Its super-hydrophilicity is associated with its excellent properties as shown in FIG. 6A, in which a random polymer having about 30 mol% of CB units has the strongest protein-repelling ability. Other polymers with higher CB content are easily detached from the PP surface due to their high solubility in aqueous solutions. Although both PCB1-28 and PCB1-37 are water insoluble, higher CB content can improve their rejection of non-specific protein adsorption. Furthermore, the concentration of the polymer solution is another important factor affecting the coating amount of the zwitterionic polymer and thus the antifouling ability. As shown in fig. 6B, as the polymer concentration increased from 0.03 wt% to 0.5 wt%, the amount of Fg adsorbed dropped dramatically, while fouling above 0.5 wt% reached a relatively low level of less than 15% of saturation compared to the uncoated substrate. In addition, both PCB1-37 and PCB2-37 polymers showed similar anti-fouling performance (FIG. 4).

The stability of the coating of CB polymer (about 30 mol% CB units) was confirmed by comparing the polymer thickness of the CB coated gold chips before and after soaking in PBS (1 ×, ph7.4) under dry conditions using a spectroscopic ellipsometer. The thickness of the modified CB polymer layer on the gold substrate before soaking was calculated to be 29.38 ± 1.10 nm. When the film thickness was maintained at 28.80 ± 0.73nm, the value did not change significantly after soaking the substrate in PBS for two months.

Adsorbed proteins from 100% blood proteins and serum. The interaction between blood components and biological materials may trigger a series of subsequent complex biological reactions including protein adsorption, platelet adhesion/activation, blood coagulation and thrombosis. Rapid non-specific adsorption of plasma proteins has been considered as the first event that occurs on the surface of biological materials during blood/material interactions. Due to the fact thatHere, it is important to assess the adsorption of major plasma proteins (e.g. fibrinogen, albumin and γ -globulin) to understand the blood compatibility of biological materials. Importantly, this evaluation often used a single protein solution at 10% dilution, but this test condition was far from the actual blood environment. As described herein, 100% single blood protein and 100% human serum were used. The amount of non-specific protein adsorption was determined by means of a Micro BCA protein assay kit.

In fig. 7A to 7D, for CB polymer coated surfaces, the adsorption capacity at 10% and 100% concentration was significantly reduced for different types of proteins and undiluted serum compared to the uncoated surface, indicating that the zwitterionic CB polymer coating can impart ultra-low fouling capability to hydrophobic hydrocarbon-based surfaces. Although commercial 96-well plates with ultra-low attachment surfaces can reject protein adsorption in single protein solutions of lower (10%) concentration, there is still about 50% protein adsorption when the protein concentration is increased to 100% (fig. 7A and 7B). Importantly, although commercially available surfaces may reject adsorption of single blood proteins at 10% concentration, they completely lost their anti-fouling ability after immersion in 100% human serum or 100% gamma-globulin environments (fig. 7C and 7D).

Cell adhesion. NIH3T3 cells were able to adhere, proliferate and migrate on normal Tissue Culture Polystyrene (TCPS) plates. After seeding them onto the surface of TCPS plates partially coated with zwitterionic CB copolymer, cells will gradually adhere and spread on the TCPS surface. In contrast, most cells will remain spherical and will not adhere to the CB copolymer surface. After 72 hours of culture and gentle rinsing with fresh medium, no adherent cells were observed on the CB copolymer coated surface, whereas cells proliferated on the normal TCPS surface and reached secondary confluency with very clear boundaries (fig. 8). For fibroblasts, adhesion is essential for maintaining multicellular structure and function, which is important for subsequent proliferation and migration. Fibronectin plays a major role in the adhesion of many cell types, and the adsorption of fibronectin will directly affect the adhesion of cells to the substrate surface. The superhydrophilic zwitterionic polymer surface has excellent antifouling ability against non-specific proteins, including fibronectin. Thus, the CB polymer coating completely prevented fibroblast adhesion.

Surface functionalization. To further evaluate the ability and versatility of CB random copolymer modified 96-well polystyrene plates to conjugate biomolecules, Fg and HSA were covalently immobilized via EDC/NHS coupling chemistry, respectively. Antibody detection is then performed via an enzymatic color reaction. Typically, anti-Fg conjugated with HRP is added to each well and ensures adequate contact with the surface. The extent of the color reaction may indirectly indicate the amount of antibody detected. The unactivated CB surface will retain anti-fouling ability without any attached biomolecules on the surface, resulting in no subsequent antibody detection (fig. 9, Fg unactivated). There was also no specific antibody-antigen induced binding for the surface that was covalently bonded to HSA but contacted with anti-Fg (fig. 9, HSA activated and HSA not activated). The degree of surface functionalization can be adjusted by varying the EDC/NHS concentration and the pH of the antibody conjugation buffer. Previous studies have shown that antifouling capability is maintained even if the CB surface is conjugated to a biomolecule moiety. Therefore, such CB polymer modified 96-well plates are promising for the detection of biomolecules in complex media, including serum, plasma and blood.

Example 1 describes the preparation, characterization and use of representative zwitterionic/hydrophobic copolymers of the present invention.

Zwitterionic/photoreactive copolymers

In another embodiment, the present invention provides a copolymer having zwitterionic groups and photoreactive groups, each group pendant to the backbone of the copolymer. The pendant zwitterionic groups impart low fouling and functionalizable properties to surfaces treated or coated with the copolymer. The pendant photoreactive groups serve to facilitate the binding (i.e., covalent coupling) of the copolymer to the surface treated with the copolymer. The relative amounts of zwitterionic groups and photoreactive groups pendant from the copolymer backbone can be adjusted via copolymer synthesis to achieve a desired degree of low contamination and functionalization and binding of the copolymer to the surface, each of which can be tailored depending on the nature (e.g., composition) of the surface to be treated or coated.

Biological contamination of implanted, blood-contacting medical device surfaces remains a serious problem with adverse biological reactions. Medical grade polyvinyl chloride (PVC) materials, particularly as blood contact tubes, have been in use on the market for decades. However, they still face serious biological contamination problems during clinical use. The properties indicate that low and high wettability surfaces are key to inhibiting surface biofouling and changes in blood composition caused by non-mild interactions.

Medical grade polyvinyl chloride (PVC) tubing is resistant to a variety of chemicals, solvents, corrosion, and has a long life expectancy and is also resistant to most sterilization methods. The extremely smooth surface of PVC provides maximum fluid flow characteristics, thereby reducing fouling that can lead to non-specific blood protein adsorption and bacterial growth. It must be noted, however, that most commercially available medical grade PVC tubes incorporate plasticizers (up to 40%) to ensure mechanical flexibility and their surfaces are very hydrophobic, which may cause serious adverse reactions when in contact with human blood, including blood protein adsorption, platelet activation/aggregation, red blood cell lysis, complement activation, etc. Although medical grade PVC tubing currently on the market has been used in hospitals for blood contact applications for decades, the design of the tubing was originally designed not to avoid the above problems. Clearly, medical grade hydrophobic PVC based materials have shown severe thrombin generation, complement activation, etc. Thus, there is a pressing need for compositions and methods for rendering medical grade PVC pipe surfaces biocompatible.

Hydrophilic biomimetic (bioinspired) antifouling materials have been used for surface coating of medical devices for decades. As a unique zwitterionic material, poly (carboxybetaine) (PCB) showed no detectable protein adsorption to undiluted human serum or plasma ((S))<0.3ng/cm²) And is widely reported in a wide range of biomedical applications as not to elicit adverse biological reactions, which exceed the performance of conventional hydrophilic or amphiphilic polymers (e.g., PEG). The Carboxyl Betaine (CB) group having an inner salt structure and having super hydrophilicity and charge neutrality may form a layerStrongly bound water molecules that cannot be displaced by biologically active substances, completely inhibiting non-specific interactions between blood components and the tube surface. Nevertheless, the direct stabilization of superhydrophilic CB polymers on the surface of commercially available hydrophobic products by simple and efficient methods to achieve practical clinical applications remains a challenge.

In some embodiments, the zwitterionic/photoreactive copolymer has superhydrophilic Carboxybetaine (CB) units and hydrophobic/photosensitive N- (4-benzoylphenyl) acrylamide (BPAA) units. In some of these embodiments, the zwitterionic/photoreactive copolymer has formula (III):

wherein

R₁And R₂Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₃and R₄Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.10 to about 0.90 mol%;

a + b is 1.0; and

represents the copolymer end groups.

In some of these embodiments, R₁And R₂Independently selected from the group consisting of hydrogen and methyl.

In some embodiments, R₃And R₄Independently selected from the group consisting of hydrogen and C1-C3 alkyl.

In some embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, n is 2 or 3.

In some embodiments, m is 1, 2, 3, 4, 5, or 6. In some embodiments, n is 1 or 2.

In some embodiments, a is from about 0.70 to about 0.90 mol%. In some embodiments, a is about 0.80 mol%.

In some embodiments, b is from about 0.10 to about 0.30 mol%. In some embodiments, b is about 0.20 mol%.

In some embodiments, R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, and X is NH and Y is NH.

In other embodiments, R₁Is hydrogen or methyl, R₂Is hydrogen or methyl, R₃And R₄Is methyl, X is O, and Y is O.

In further embodiments, R₁Is hydrogen, R₂Is hydrogen, R₃And R₄Is methyl, X is NH, Y is NH, n is 3, and m is 1. In some of these embodiments, a is about 0.80 mol% and b is about 0.20 mol%.

The following is a description of the preparation, characterization and use of representative zwitterionic/photoreactive copolymers of the invention and their use in the compositions and methods of the invention.

The present invention provides a surface modification strategy that directly imparts superhydrophilic and antifouling capabilities to commercially available hydrophobic medical grade PVC tubing via a simple and effective dip coating process. This strategy is achieved by covalently grafting zwitterionic Carboxybetaine (CB) copolymers to the inner surface of PVC pipes by light induced conjugation and self-crosslinking. CB copolymer coated commercial medical grade PVC tubing (Streamline air System Set, medisys Corporation, MA, USA) showed high surface wettability and ultra low contamination to 100% human serum.

FIG. 10A is a schematic of the process of the present invention: the CB copolymer was covalently grafted to the inner surface of medical grade PVC tubing by light induced conjugation and crosslinking. Representative photoreactive CB copolymers (fig. 10B) were prepared by conventional free radical polymerization, medical grade PVC tubing coated with synthetic polymer and stabilized under UV irradiation. The surface grafted with PCB copolymer by dip coating showed better resistance to non-specific protein adsorption of 100% human serum compared to the uncoated tubes. Therefore, this surface modification strategy is very promising for improving the biocompatibility of current commercial medical grade PVC tubing.

Amphiphilic random copolymers were synthesized by conventional free radical polymerization methods using AIBN as a thermal free radical initiator. The viscosity of these reaction solutions gradually increased as polymerization proceeded at 65 ℃ indicating the conversion of monomer to copolymer. From¹Calculation of the integral value of the characteristic peak in the H-NMR spectrum gives the unit fraction of each monomer. All functional units being present by¹H NMR was verified, where the unit fraction of each monomer was obtained from the integral value of the characteristic peaks: CB unit is 3.82ppm (-CH)₂-, 2H), BPAA units from 6.80 to 7.85ppm (benzophenone-H, 9H). The chemical structure of this polymer is shown in fig. 10B. Both the hydrophilic CB monomer and the hydrophobic/photosensitive BPAA in the polymer chain are randomly distributed with an overall composition approximately equal to that of the monomer feed solution. These results are highly compatible with other random copolymers containing phosphorylcholine groups, which have been widely used for surface modification of biomedical devices.

UV light transmission tests showed that the PVC pipe had 50% light transmission at a wavelength of 312nm, which is the optimal irradiation wavelength for the benzophenone group (fig. 11A). Therefore, the physically adhered polymer on the inner surface can be easily stabilized by applying external UV light. The results show that the polymer is very sensitive to UV light (312nm) and the absorption spectrum varies between 300 and 350nm, indicating that photo-induced covalent bonding and self-crosslinking to the surface of the PVC pipe occurs (fig. 11B).

Hydrophilicity is one of the key properties of the grafted surface of the PCB. Water contact angles under dry conditions were measured directly on coated and uncoated medical grade PVC tubing. The results show that on the bent PVC pipe (fig. 12A), the water contact angle on the PCB coated pipe is much smaller than on the uncoated pipe. Generally, the water contact angle of a PCB coated flat PVC surface is around 10 °, while the water contact angle of an uncoated flat PVC surface is greater than 85 ° (fig. 12B). Thus, grafting of the PCB copolymer converts the hydrophobic PVC pipe surface to a superhydrophilic surface.

To bring our technique closer to clinical use, X-ray photoelectron spectroscopy (XPS) was used to verify the presence and stability of PCB polymers. The Binding Energy (BE) was corrected using the peak of C1s at 285eV as a reference. XPS results showed that the PCB coated surface had the same N1s peak as the original PCB polymer (fig. 13A to 13D) and obtained an unchanged atomic composition (steady) even after 3 weeks of storage in the dry state and at room temperature (table 1).

Table 1 surface atomic composition of different samples.

The results show successful grafting of PCBs onto the inner surface of commercially available medical grade PVC tubing with excellent stability. Interestingly, a peak of zinc (Zn 2p) was also observed from the measured spectra, indicating the presence of ZnO, which is commonly coated on medical grade PVC surfaces to enhance antimicrobial capability.

Assessment of biological contamination is crucial to assessing the clinical application potential of current design strategies. Protein adsorption is the major event that occurs on the surface of biological materials in contact with biological environments. In fig. 14, the amount of serum protein adsorbed on the CB polymer coated tubes was significantly reduced compared to the uncoated tubes, indicating that the zwitterionic CB polymer coating had high anti-fouling capability. Importantly, there was no difference in the fouling levels of PCB coated tubes stored for 1 week and 3 weeks. This further demonstrates the stability of the PCB coating and consistency with XPS results. All results show that representative PCB copolymers can impart superhydrophilicity and durable stain resistance to medical grade PVC tubing.

In summary, in one aspect, the present invention provides a functional random amphiphilic zwitterionic copolymer having a superhydrophilic Carboxybetaine (CB) unit and a hydrophobic/photosensitive N- (4-benzoylphenyl) acrylamide (BPAA) unit, which is useful as a surface coating material. CB random copolymer modified medical grade PVC tubing has significantly improved antifouling properties compared to commercially available uncoated medical grade PVC tubing with ultra-low adhesion surface. Surface coating via a dip coating process is advantageously simple and effective for large scale applications. Its utility is further verified by its ability to achieve non-invasive coating and long-term persistence. Therefore, the technique is expected to be used in a wide range of medical and engineering applications, especially in medical grade PVC tubing.

Example 2 describes the preparation, characterization and use of a representative zwitterionic/photoreactive copolymer of the present invention.

In a related embodiment, the present invention provides a surface modification strategy to directly inhibit or prevent plasticizer leaching from a surface (e.g., a plastic surface, such as a polyvinyl chloride surface (PVC) or a Polyurethane (PU) surface), such as commercially available hydrophobic medical grade PVC or PU tubing, via a simple and effective method. In one embodiment, this strategy is achieved by covalently grafting zwitterionic Carboxybetaine (CB) copolymers to the inner surface of PVC pipes by light-induced conjugation and self-crosslinking.

Surface modification of commercially available polyvinyl chloride (PVC) medical devices with the zwitterionic copolymers of the present invention improves the biocompatibility of the PVC and prevents migration of plasticizers (e.g., phthalates such as di-2-ethylhexyl phthalate (DEHP)) from the PVC to the patient under varying shear stresses.

The following is a description of the preparation, characterization and use of representative zwitterionic/photoreactive copolymers of the present invention to impart biocompatibility and inhibit leaching of plasticizers on polyvinyl chloride.

Biological contamination of implanted, blood-contacting medical device surfaces remains a serious problem with adverse biological reactions. Medical grade polyvinyl chloride (PVC) materials, particularly as blood contact tubes and containers, have been used for decades. However, these materials face (a) problems associated with biological contamination (e.g., platelet activation, complement activation, and thrombin generation) and (b) leaching problems of toxic plasticizers in clinical applications. The invention provides a surface modification method which can obviously prevent blood protein pollution, human platelet activation and complement activation on medical PVC materials sold in the market under various dynamic disturbances. Surface coatings can be obtained via simple and efficient dip coating using biocompatible polymers consisting of zwitterionic Carboxybetaine (CB) groups and photosensitive crosslinking groups, followed by light irradiation. Such biocompatible polymers with adjustable functional groups can be routinely manufactured on any scale and impart superhydrophilic and antifouling capabilities to commercial PVC materials. In addition, the polymer effectively prevents leaching of toxic plasticizers from commercially available medical grade PVC materials. This technique is readily applicable to many other medical devices requiring biocompatible surfaces.

Medical grade polyvinyl chloride (PVC) has been used in flexible medical products because of its resistance to most chemicals, solvents and sterilization methods, and its low cost. Although these products have passed initial critical toxicological, biological and physiological tests, medical grade PVC-based materials continue to receive increasing criticism due to the emergence of severe blood protein adsorption, platelet activation/aggregation, red blood cell lysis, thrombin generation, complement activation, etc., as adverse interactions between non-biocompatible PVC and blood components can occur. Blood protein adhesion plays an important role in determining the biocompatibility of the material, which is considered to be a major event in triggering subsequent adverse reactions. Thus, imparting a biocompatible surface to medical grade PVC will effectively reduce biofouling buildup.

In fact, about 30% of all plastic-based disposable medical devices used in hospitals are generally made of flexible PVC physically mixed with up to 40 wt% of plasticizers (e.g. phthalates) to ensure its mechanical flexibility as blood contact tubes, containers, etc. Importantly, plasticizers leach from PVC materials into patients to varying degrees upon contact with blood, potentially causing serious adverse health effects (e.g., nephrotoxicity, endocrine toxicity, reproductive diseases, neurotoxicity, hepatotoxicity, and cardiotoxicity) in some patient populations. Therefore, many studies on non-toxic plasticizers and non-migratory alternative plasticizers have been reported. However, due to the lack of comprehensive assessment of their long-term health effects, functional effectiveness and cost considerations, most medical grade PVC materials currently on the market are still prepared using conventional methods, thus facing the leaching problem of toxic plasticizers. It is therefore desirable to modify medical grade PVC products with biocompatible materials that effectively eliminate adverse reactions caused by biological contamination and prevent migration of toxic plasticizers, while not affecting the significant properties of the PVC base such as inertness, sterilizability and flexibility.

Current methods to improve the biocompatibility of medical grade PVC materials include the introduction of hydrophobic ingredients (e.g. silicone derivatives and polytetrafluoroethylene), grafting hydrophilic anti-fouling materials (e.g. allylamine, poly (acrylamide), polyethylene glycol) and modifying the surface nano/micro structure. However, none of them clearly demonstrated the ability to simultaneously address both non-biocompatibility and plasticizer leaching problems. Clearly, hydrophobic materials containing a fouling release moiety (e.g., a silicone derivative) may exhibit undesirable biological reactions, such as platelet/complement activation, during adhesion release cycles. Importantly, blood proteins have a higher affinity for adhesion to hydrophobic surfaces, and they exhibit less organized secondary structures when adsorbed onto hydrophobic surfaces than when adsorbed onto hydrophilic surfaces.

Hydrophilic anti-fouling materials have been used for decades for surface coating of medical devices, including zwitterionic polymers, poly (ethylene glycol), poly (hydroxy functional acrylates), poly (2-oxazoline), poly (vinyl pyrrolidone), poly (glycerol), peptides and peptoids. As a unique zwitterionic material, poly (carboxybetaine) (PCB) showed no detectable protein adsorption to undiluted human serum or plasma ((S))<0.3ng/cm²) And is widely reported in a wide range of biomedical applications as not to induce adverse biological reactions, which exceed the performance of conventional hydrophilic or amphiphilic polymers. Carboxybetaine (CB) groups with super-hydrophilicity and charge neutrality can form a layer of strongly bound water molecules, thereby completely inhibiting non-specific interactions between blood components and the tube surface. Nevertheless, the direct stabilization of superhydrophilic CB polymers on commercially available hydrophobic tube surfaces by simple and efficient methods to achieve practical clinical applications is a huge challenge. Furthermore, surface modification of plasticized PVC is one of the most commonly studied strategies in order to prevent plasticizer leaching,of which surface cross-linking is the most successful technique.

In one embodiment, the present invention provides a surface modification process of carboxybetaine copolymer (PCB) aimed at directly imparting superhydrophilic and antifouling capability to commercially available hydrophobic medical grade plasticized PVC tubing (Streamline air System Set, medisys Corporation, MA, USA), as well as low plasticizer migration from PVC tubing. PCB copolymers with tunable functional groups (i.e. zwitterionic carboxybetaine groups and photosensitive crosslinking groups) were prepared by free radical polymerization. Medical grade plasticized PVC tubing was dip coated with this polymer and then Ultraviolet (UV) light at a wavelength of 312nm was applied to the coated surface to stabilize the coated PCB polymer layer and prevent migration of the plasticizer through surface crosslinking. The stability and non-cytotoxicity of the PCB polymers were confirmed by X-ray photoelectron spectroscopy (XPS) and examined based on ISO10993-5 guidelines, respectively. The wettability and antifouling capacity of the coated PVC surface, the level of platelet activation and the level of complement activation were directly compared to those of the uncoated surface. Clearly, the antifouling ability to 100% human serum under different dynamic perturbation/shear stresses was verified using Surface Plasmon Resonance (SPR) and effectively prevented plasticizer migration of PVC tubing. The method demonstrates the biocompatibility of the PCB copolymer under different dynamic perturbations and the ability to inhibit plasticizer leaching. In addition, these materials and surface modification strategies are also applicable to other medical devices.

Synthesis of copolymer. Photoreactive amphiphilic random copolymers were synthesized by conventional radical polymerization methods using AIBN as a thermal radical initiator. The process ensures that such polymers with tunable functional groups can be routinely produced on any scale, particularly for industrial scale-up. The viscosity of these reaction solutions gradually increased with polymerization at 65 ℃ indicating the conversion of monomer to copolymer. The chemical structure of the poly (CBAA-co-BPAA) (PCB) polymer is shown in FIG. 15A. The CBAA group has super-hydrophilicity and complete charge neutrality, and can effectively inhibit the adhesion of blood proteins, cells and bacteria. The photosensitive group (BPAA) can stabilize the polymer on the PVC surface by light-induced crosslinking, thereby further preventing plasticizer migration.All functional units being present by¹H NMR verification, in which the unit fraction of each monomer is obtained from the integral value of the characteristic peak: CB unit is 3.82ppm (-CH)₂-, 2H), BPAA units from 6.80 to 7.85ppm (benzophenone-H, 9H). The hydrophilic CB monomers and hydrophobic/photosensitive BPAA in the polymer chain are randomly distributed with a total composition approximately equal to the composition of the monomer feed solution (table 2). The chemical structures of poly (CBAA-co-BPAA-co-NB acrylamide) (PCB-NB) and poly (PEGMA-co-BPAA) (PPB) are shown in FIGS. 15B and 15C, respectively.

TABLE 2 characteristics of representative synthetic copolymers^a

^a[ monomer]0.5 mol/L. The initiator for all polymers was AIBN. The polymerization temperature was 65 ℃.^bFrom CD₃In OD¹H NMR spectrum determination.^cMolecular weight by GPC in methanol/water 7/3, [ LiBr ═]10mmol/L and poly (ethylene oxide) standards. M_wAnd M_nRespectively represent the weight average molecular weight and the number average molecular weight.^dFor each polymer sample, solubility was determined at a concentration of 10mg/mL and was described as soluble (+) or insoluble (-) -at 25 ℃.^eThe molar ratio in the feed and copolymer was PEGMA/BPAA.

Surface characterization. UV light transmission tests showed that the commercial PVC pipe had 50% light transmission at a wavelength of 312nm (fig. 16A and 16B), which is the optimal wavelength of irradiation for the benzophenone group in the PCB copolymer. Thus, application of external UV light will stabilize the PCB polymer on the inner surface of the tube. The results in (FIG. 16C) show that the polymer is very sensitive to UV light (312nm) with a change in absorption spectrum from 300 to 350nm, indicating photo-induced covalent bonding and self-crosslinking of the PVC pipe surface. Hydrophilicity is one of the key properties of the grafted surface of the PCB. Water contact angles under dry conditions were measured directly on coated and uncoated medical grade PVC tubing. The water contact angle on the PCB coated tube was much smaller than on the uncoated tube (fig. 16D). In general, PCB coated flat PVC surfaces have water contact angles around 10 ° while uncoated flat PVC surfaces have water contact angles greater than 85 °. Thus, grafting of the PCB copolymer converts the hydrophobic PVC pipe surface to superhydrophilic.

Stability of the coating. The FDA in the united states requires that containers that come into contact with blood and blood components have no paint leaching problems. To make this technique more practical, X-ray photoelectron spectroscopy (XPS) was used to verify the presence and stability of PCB polymers. The Binding Energy (BE) was corrected using the peak of C1s at 285eV as a reference. XPS results showed that the PCB coated surface had the same N1s peak as the original PCB polymer (fig. 16E to 16H) and achieved an unchanged atomic composition even after 3 weeks of storage in the dry state and at room temperature. Thus, PCBs were successfully grafted onto the inner surface of commercially available medical grade PVC tubing with excellent stability. A peak in zinc (Zn 2p) is also observed from the measured spectra, indicating the presence of zinc compounds (e.g., ZnO) that are typically coated on the surface of medical grade PVC to enhance antimicrobial capability. In addition, the stability of the PCB in humid conditions was also evaluated. Commercial tubes were coated with fluorescently labeled PCB polymer (PCB-NB) and soaked in PBS/100% human plasma at 37 ℃. The amount of leached PCB-NB in the solution was evaluated by analyzing UV/Vis spectra at 590nm wavelength. The results show that no absorption peak of the PCB polymer was observed after soaking in PBS or 100% human plasma at 37 ℃ for 24 hours, indicating the stability of the PCB polymer under humid conditions.

Assessment of biological contamination. Protein adsorption plays an important role in biocompatibility assessment, which is a major event occurring on the surface of biomaterials in biological environments. Importantly, protein adsorption behavior depends on the surface characteristics of the biomaterial and various shear stresses. Both albumin and fibrinogen showed stronger binding affinity and less organized secondary structure to hydrophobic alkyl surfaces than to hydroxyl-terminated hydrophilic surfaces, with greater effect on albumin observed. With increasing shear rate, a decrease in plasma protein adsorption to the polyurethane surface was observed. Described herein are the use of Surface Plasmon Resonance (SPR) biosensors and Micro, respectively, with adjustable flow ratesAdsorption behavior of plasma proteins on the zwitterionic carboxybetaine polymer surface under dynamic and static conditions of the BCA protein assay kit.

The amount of non-specifically adsorbed proteins on the uncoated (SPR chip coated with PVC) and PCB coated (SPR chip coated with PVC first layer and PCB polymer second layer) surfaces was assessed using SPR before serum injection and after buffer washing (fig. 17A to 17D). The sharp increase after serum injection is due to the change in the bulk refractive index. To compensate for the loss of SPR surface sensitivity due to the coated polymer layer, the response of the sensor to protein adsorption was calibrated at different polymer thicknesses. The protein adsorption on the PCB-coated surface was 4.3, 3.3, 3.6 and 4.4ng/cm at four different flow rates of 10, 40, 100 and 200. mu.L/min, respectively²And the protein adsorption amounts on the uncoated surface were 78.7, 85.9, 81.9, and 81.4ng/cm²(FIG. 17E). The results show that the PCB coated surface has ultra low contamination compared to the uncoated surface, regardless of shear stress, under dynamic conditions (<5.0ng/cm²) Capability and<10% contamination and the results were consistent with those of the Micro BCA protein assay kit under static conditions (fig. 17F). Furthermore, there was no difference in the fouling levels of PCB coated tubes stored in PBS for 1 week and 3 weeks. This further demonstrates the stability of the PCB coating and consistency with XPS results under dry conditions. Therefore, the zwitterionic PCB copolymer can endow the medical grade PVC pipe with super-hydrophilicity and durable anti-fouling capability.

PCB Polymer toxicity. The cytotoxicity of PCB polymers was examined according to ISO10993-5 guidelines. The morphology of NIH3T3 cultured in the eluent of the PCB coated tubes or the medium containing the dissolved PCB polymer was observed and compared to the control. As shown in figure 18A, the morphology of NIH3T3 cultured in the eluate from the PCB-coated tubes was not different from the morphology of the uncoated tubes and the control samples in normal cell culture medium. In contrast, cells lost the original spindle shape after incubation in latex eluents of toxic materials used as positive controls. Apparently, in cultures containing solubilized PCB polymers compared to controlsThe morphology of the cells cultured in the medium was also unchanged (fig. 18B). Thus, PCB polymers are not toxic according to ISO10993-5 guidelines. In addition, cytotoxicity induced by different concentrations (0.00125, 0.0025, 0.01 and 0.1mg/ml) of PCB polymer was assessed by leakage of LDH into the culture medium. A positive correlation between the absorbance value at 560nm and the relative LDH level was used to assess cell membrane damage caused by the polymer. There was no significant difference between the LDH release of cells cultured with the PCB-containing medium and the culture under normal conditions. In contrast, cells cultured with 0.20 vol.% Tween 20 medium solution released far higher levels of LDH than the others (fig. 18C). These results indicate that the PCB polymer does not exert cytotoxic effects on the membrane of living cells.

Effect of PCB polymers on platelet quality. The quality of platelets in PCB-coated and uncoated PVC surfaces was compared to verify the potential of PCB polymers for blood-contacting medical devices. The level of platelet activation and binding affinity to Von Willebrand Factor (VWF) are the most critical parameters for assessing the ability of platelets to clot, directly reflecting the quality of the platelets. p-selectin (CD62) is a glycoprotein and a well-described marker of platelet activation. During platelet activation, p-selectin is transferred from the intracellular granules to the outer membrane. The level of platelet activation of the PCB-coated surface was much lower than that of the uncoated PVC surface, indicating that platelet activation was effectively inhibited by using the PCB polymer (fig. 19A). The functional testing of platelets is the most critical parameter for assessing the ability of platelets to clot. VWF, an adhesive plasma glycoprotein, plays an important role in the formation of platelet embolisms under physiological conditions, allowing circulating platelets to adhere to and embolize sites of vascular injury. Higher Mean Fluorescence Intensity (MFI) (fig. 19B) correlates with higher binding affinity to VWF. Platelets of the PCB-coated surface had a higher binding affinity to VWF within 0 to 168 hours than platelets of the uncoated surface, which became more apparent after 168 hours. Annexin V is commonly used to detect apoptotic cells by its ability to bind phosphatidylserine, which is a hallmark of platelet apoptosis when it occurs in the outer plasma membrane lobes. Due to the fact thatThus, higher binding of annexin V indicates increased apoptosis and decreased viability. As shown in fig. 19C, platelets on the PCB-coated hydrophilic surface had lower annexin V binding levels than platelets on the uncoated surface, indicating that the PCB-modified surface can effectively maintain platelet viability. Healthy inactivated platelets are biconvex disk-like structures with a maximum diameter of 2-3 μm. As the health of platelets deteriorates, a change in shape from a disc to a sphere (disc-to-sphere conversion) occurs. Morphological Score (MS) was used as a simple indicator of platelet health and was defined as 4 × (dish%) +2 × (sphere%) + (branch%). The MS values of platelets on the PCB-coated surface were much higher than those on the uncoated PVC surface (fig. 19D). Thus, the PCB polymer on the hydrophobic PVC surface can effectively prevent the deterioration of platelet quality, which makes the PCB polymer promising for the manufacture of blood contact devices.

Plasticizer leaching. Phthalates, especially di-2-ethylhexyl phthalate (DEHP), are the most commonly used plasticizers for commercial PVC because of their ease of plasticization and processing and competitive cost. However, despite the toxicity of DEHP as demonstrated by many studies, PVC materials containing DEHP remain controversial and have not been banned for biomedical applications. Current approaches to eliminating the toxicity of plasticized materials include (a) inhibiting the leaching of DEHP plasticizers by chemical or physical surface modification, (b) developing alternatives to DEHP (e.g., adipates, azelates, citrates, and trimellitates), and (c) creating plasticizer-free PVC alternative polymers (e.g., silicones, polyurethanes, and polyolefins). Inhibition of DEHP leaching may be the most adequate method of evaluation requiring long-term and comprehensive evaluation. It is clear that surface crosslinking of PVC materials is the most successful technique for effectively preventing DEHP leaching at the present stage. DEHP has three spectral bands around 210nm, 225nm and 275 nm. The absorption values at 275nm wavelength of the coated and uncoated PVC pipes were compared as described herein. The results in fig. 20 show that the level of leached DEHP from the PCB coated PVC pipe is significantly lower than from the uncoated pipe, about 12% of the uncoated pipe at 24 hours. This demonstrates that surface grafted photoreactive PCB polymers effectively prevent DEHP migration and are expectedFor eliminating the toxicity of plasticized PVC materials. Photo-induced surface cross-linking of PCB polymers may be critical to stabilize the PCB polymers and prevent plasticizer migration at the PVC surface.

Complement activation. The complement system, which comprises a series of more than 20 proteins circulating in blood and interstitial fluid, is a major contributor to the innate immune system that can eliminate foreign cells and organisms either by direct lysis or by recruitment of leukocytes that promote phagocytosis. The complement system is activated by three pathways: the classical pathway, the alternative pathway and the lectin pathway. The surface of artificial biomaterials triggers the alternative pathway mainly by adsorbing the metastable complement protein C3b, which further initiates the entire complement cascade to induce the formation of terminal complement complexes that activate leukocytes and induce inflammatory responses (sC5 b-9). Thus, the amount of sC5b-9 reflects the ability of the biological material to stimulate the complement system. Importantly, oxidation of both the terminal hydroxyl (-OH) group and the poly (ethylene glycol) (PEG) chain strongly activates the complement system via an alternative pathway, and thus the copolymer poly (PEGMA-co-BPAA) (PPB) would be a complement system activator (positive control). The results in fig. 21 show that serum contacted with PCB coated or uncoated medical grade PVC material had lower levels of terminal complement complex (sC5b-9) than serum contacted with PPB coated material, indicating that the PCB polymer had lower affinity for the activated complement system than the PPB coated surface. Furthermore, the complement cascade was not completely inhibited on neither PCB coated nor uncoated surfaces, and there was no significant difference between them. This is because additional complement activation is triggered at the interface between serum and air (plasma proteins denatured and/or conformationally altered), which is amplified and cannot be terminated once triggered. For a PPB coated surface, the activation caused by the PPB copolymer is much more severe than the activation caused by the serum/air interface, and therefore the overall level of activation is high. Thus, these results indicate that the PCB polymer is not a complement-activating biomaterial.

In summary, in one embodiment, the present invention provides a functional random type amphiphilic zwitterionic copolymer comprising super hydrophilic Carboxybetaine (CB) units and hydrophobic/photosensitive N- (4-benzoylphenyl) acrylamide (BPAA) as a surface coating material. The CB random copolymer modified medical grade PVC pipe can obviously prevent blood protein pollution, human platelet activation and complement activation under different dynamic disturbances. Such surface coatings can be applied simply and efficiently on a large scale via a dip coating process followed by irradiation with UV light. Its utility is further verified by its ability to achieve non-invasive coating and long-term persistence. In addition, the polymer effectively prevents leaching of toxic plasticizers from commercially available medical grade PVC materials. Therefore, the method simultaneously solves the problems of non-biocompatibility and plasticizer leaching of the PVC material. The method is readily applicable to many other medical devices requiring biocompatible and additive leaching inhibiting surfaces.

Example 3 describes the preparation, characterization and use of representative zwitterionic/photoreactive copolymers of the present invention to prevent leaching of plasticizers from plastic surfaces.

Zwitterionic/hydrophobic/photoreactive copolymers

In a further embodiment, the present invention provides a copolymer having a zwitterionic group, a hydrophobic group, and a photoreactive group, each group being pendant from the backbone of the copolymer. The pendant zwitterionic groups impart low fouling and functionalizable properties to surfaces treated or coated with the copolymer. The pendant hydrophobic groups serve to facilitate binding of the copolymer to a surface treated with the copolymer (e.g., via hydrophobic-hydrophobic interactions). The pendant photoreactive groups serve to facilitate crosslinking of the copolymer on the surface treated with the copolymer. The relative amounts of the zwitterionic groups, hydrophobic groups, and photoreactive groups pendant on the copolymer backbone can be adjusted via copolymer synthesis to achieve the desired degree of low contamination and functionalization and binding of the copolymer to the surface, each of which can be adjusted depending on the nature (e.g., composition) of the surface to be treated or coated.

In some of these embodiments, the present invention provides zwitterionic copolymers having superhydrophilic Carboxybetaine (CB) units, hydrophobically bound N-Butyl Methacrylate (BMA) units, and hydrophobic water/photosensitive N- (4-benzoylphenyl) acrylamide (BPAA) units for use as surface coating materials for platelet storage bags, methods of coating surfaces of platelet storage bags with the copolymers, and platelet storage bags coated with the copolymers.

Platelets are unique blood components that play a vital role in hemostasis, thrombosis, inflammation, and wound healing. Platelet-based therapy, i.e., platelet infusion, is an effective method of treating bleeding in patients suffering from thrombocytopenia or platelet dysfunction. Importantly, platelets stored in vitro have a shelf life of only half (4-7 days) of their in vivo blood flow (8-10 days) under current standard conditions. Therefore, there is a need to improve platelet storage conditions to extend their shelf life, thereby alleviating the growing demand for platelets. The shelf life of platelets is strongly affected by two reasons: (i) bacterial contamination and (ii) Platelet Storage Lesions (PSLs). The risk of bacterial contamination can be reduced by strict aseptic collection and highly sensitive bacterial diagnostics, while the main limiting factors PLS are strongly influenced by storage conditions, including vessel, buffer composition, temperature, respiratory gas exchange efficiency, pH reduction, lactic acid accumulation, nutrient consumption, etc. Although many attempts have been made, including novel storage medium additives, refrigeration and lyophilization, for practical applications, the current standard platelet preservation of polyvinyl chloride is to shake (agitatating) platelets in hydrophobic (e.g., polyvinyl chloride) storage bags at room temperature (20-24 ℃). In fact, high quality platelet storage has never been extended to 8 days. One of the keys to mass loss is the adverse interaction between platelets and the surface of the non-biocompatible storage bag, including unrecoverable plasma protein denaturation, platelet activation and biofilm formation on hydrophobic plasticized PVC materials. Clearly, medical grade hydrophobic PVC based materials also show severe thrombin generation and complement activation. Thus, rendering a hydrophobic surface of a storage bag biocompatible remains a major challenge.

Currently, the biocompatibility of platelet bags is improved from two aspects: (i) modification of surface nano/micro structures/patterns with mixtures of soil release materials (e.g. silicone derivatives)To impart superhydrophobicity, and (ii) grafting a neutral biomimetic anti-fouling material (e.g., a zwitterionic polymer, poly (ethylene glycol), or poly (2-oxazoline)) to impart superhydrophilicity to the surface. Importantly, it remains suspected that an anti-fouling surface, as verified by traditional methods involving a washing process, is a biocompatible surface, since hydrophobic materials, particularly those containing a fouling release moiety (e.g., silicone), may eventually exhibit low biofouling but severe adverse biological reactions (e.g., platelet activation, complement activation) during the adhesion release cycle. Although platelet activation is generally considered to be a subsequent reaction to adhesion caused by fibrinogen adsorption, non-platelet/protein-adhesive surfaces do not completely prevent platelet activation. Therefore, the assessment of the amount of finally adsorbed protein/platelets is not sufficient and it is crucial to study the platelet properties in solution. For decades, hydrophilic biomimetic anti-fouling materials have been used for surface coating of medical devices. As a unique zwitterionic material, poly (carboxybetaine) (PCB) showed no detectable protein adsorption to undiluted human serum or plasma ((S))<0.3ng/cm²) And is generally reported not to elicit adverse biological reactions in a wide range of biomedical applications, beyond the performance of conventional hydrophilic or amphiphilic polymers (e.g., PEG). The Carboxybetaine (CB) group having an inner salt structure, having super-hydrophilicity and charge neutrality, can form a layer of strongly bound water molecules that cannot be substituted by bioactive substances, thereby completely inhibiting non-specific interaction between blood components and the surface of the pouch. Nevertheless, the direct stabilization of superhydrophilic CB polymers on the surface of commercially available hydrophobic products by simple and efficient methods to achieve practical clinical applications remains a challenge.

In some embodiments, the present invention provides a copolymer of formula (IV):

wherein

R₁、R₂And R₃Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

R₄and R₅Independently is- (CH)₂)_xH, wherein x is an integer from 0 to 20;

x is O or NH;

y is O or NH;

z is O or NH;

n is an integer of 1 to 20;

m is an integer of 1 to 20;

p is an integer of 0 to 20;

a is about 0.10 to about 0.90 mol%;

b is about 0.05 to about 0.95 mol%;

c is about 0.05 to about 0.95 mol%;

a + b + c is 1.0; and

represents the copolymer end groups.

In some of these embodiments, R₁、R₂And R₃Independently selected from the group consisting of hydrogen or methyl.

In some embodiments, R₄And R₅Independently selected from the group consisting of hydrogen or C1-C3 alkyl.

In some embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, n is 2 or 3.

In some embodiments, m is 1, 2, 3, 4, 5, or 6. In some embodiments, m is 1 or 2.

In some embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 3.

In some embodiments, a is from about 0.70 to about 0.90 mol%. In some embodiments, a is about 0.70 mol%.

In some embodiments, b is from about 0.05 to about 0.25 mol%. In some embodiments, b is about 0.20 mol%.

In some embodiments, c is from about 0.05 to about 0.20 mol%. In some embodiments, b is about 0.10 mol%.

In one embodiment, R₁、R₂And R₃Is hydrogen, R₄And R₅Is methyl, X is NH, Y is O and Z is NH.

In another embodiment, R₁、R₂And R₃Is hydrogen, R₄And R₅Is methyl, X is NH, Y is O, Z is NH, n is 3, m is 1, and p is 3.

In some of the above embodiments, a is about 0.70 mol%, b is about 0.20 mol%, and c is about 0.10 mol%.

The following is a description of the preparation, characterization and use of representative zwitterionic/hydrophobic/photoreactive copolymers of the present invention and their use in the compositions and methods of the present invention.

The present invention provides a surface modification strategy for CB polymers that directly imparts superhydrophilicity and antifouling capabilities to commercially available hydrophobic platelet storage bags by an extremely simple and effective dip coating technique. This was achieved by covalently grafting the CB copolymer to the inner surface of the storage bag by light induced conjugation and cross-linking (fig. 22A). The CB moiety is superhydrophilic, which makes it difficult to attach to commercially available hydrophobic platelet storage bags, with hydrophobic binding groups (n-butyl methacrylate, BMA) (e.g., 20 mol%) and photosensitive groups (benzophenone) (e.g., 10 mol%) included in the copolymer. Benzophenone groups have been widely used as photoinitiators to facilitate chemical conjugation, which under UV irradiation at 250 to 365nm produces an extraction of aliphatic hydrogens to form covalently bound diradicals. To introduce such a binding group into the copolymer, the monomer N- (4-benzoylphenyl) acrylamide (BPAA) was synthesized and then copolymerized with other monomers to form a multifunctional copolymer (FIG. 22B). Presence of functional units by¹H NMR verification, in which the unit fraction of each monomer is obtained from the integral value of the characteristic peak: CB unit is 3.82ppm (-CH)₂-, 2H), BMA unit of 1.45 to 1.63ppm (-CH)₂-, 4H), BPAA units from 6.80 to 7.85ppm (benzophenone-H, 9H). The results show that the polymer is very sensitive to UV light (312nm) and the absorption spectrum changes from 300 to 350nm, indicating that photo-induced covalent bonding occurs (fig. 22C).

The modification of the commercially available platelet bags occurs only at the inner surface, without compromising the properties of the original bag. To achieve this goal, a representative PCB polymer was dissolved in DI water at a very low concentration (0.5 wt%) and then injected into the bag with gentle shaking to ensure that all surfaces could be coated with the polymer. UV light transmission tests showed that the bag had 70% light transmission at a wavelength of 312nm, which is the optimal irradiation wavelength for the benzophenone group (fig. 23A). Therefore, the physically adhered polymer on the inner surface can be easily stabilized by applying external UV light. The copolymer coating thickness using this technique is typically around <50nm, so we do not see significant differences from the SEM image (fig. 23B). Interestingly, a textured surface was found which prevents the inner surface from clogging during heat sterilization or blood treatment. Other studies have shown that textured surfaces have more severe biofouling than smooth surfaces. Commercially available platelet bags are mainly made of PVC mixed with plasticizers (up to 40%) to ensure the inherent mechanical flexibility and breathability of the bag. Thus, the surface was briefly rinsed with ethanol in a short time (10 seconds) to reduce the leaching effect of the plasticizer on the PCB stability.

The united states Food and Drug Administration (FDA) requires that empty containers that will contact blood and blood components should not have leaching problems. Therefore, in order to bring this technique closer to clinical applications, X-ray photoelectron spectroscopy (XPS) was used to verify PCB stability. The Binding Energy (BE) was corrected using the peak of C1s at 285eV as a reference. XPS results showed that the PCB coated surface had the same N1s peak as the original PCB polymer (fig. 23C and 23D) and obtained an unchanged atomic composition even after soaking in buffer for 2 weeks (fig. 23E), which can cover the entire life cycle of the platelets. Thus, PCBs were successfully grafted onto the inner surface of commercially available platelet bags and had excellent stability. Interestingly, two silicon peaks (Si 2s and Si 2p) were observed from the measured spectra, demonstrating that the bag was treated with hydrophobic silicon-based materials with fouling release properties. This phenomenon was further confirmed by real-time monitoring of contamination of 100% human serum under dynamic conditions (fig. 24A). Hydrophilicity is one of the key properties of the grafted surface of the PCB. The water contact angle under dry conditions and the air contact angle in aqueous media were measured. All contact angles were measured directly from the photographic images. The water contact angle of the PCB-coated surface decreased significantly from 90 ° to 10 ° and the air contact angle increased significantly from 80 ° to 160 ° (fig. 23F). Thus, grafting of the PCB copolymer converts the hydrophobic surface to a superhydrophilic surface.

Despite decades of research, biological contamination on the surfaces of implanted, blood-contacting medical devices remains a serious problem. PCB coated platelet bags will be used for platelet preservation, ultimately involving direct contact with human blood proteins and platelets. Therefore, biological contamination assessment is crucial to assess the clinical application potential of current design strategies. Protein adsorption is the major event that occurs on the surface of biological materials in contact with biological environments. Proteins may exhibit different adsorption behavior under dynamic and static conditions. Thus, PCB-coated commercial platelet bags against 100% human serum were evaluated under dynamic and static conditions using Surface Plasmon Resonance (SPR) and Micro BCA protein assay kits, respectively. SPR is widely used to characterize in situ real-time interactions between polymer surfaces and proteins, limit of detection<0.3ng/cm². The SPR slide was spin coated with a commercially available platelet bag/THF solution and a PCB copolymer/DI aqueous solution in that order. The thickness of the first layer (molten PVC pouch) and the second layer (PCB polymer) was measured under dry conditions and was 18 ± 1.2nm and 20 ± 2.1 nm. Figure 24A shows the change in SPR signal due to serum adsorption as a function of time. For chips coated with fused platelet bags, the wavelength shift increased and subsequently showed a steady decreasing trend as flow of 100% human serum was continued, indicating that XPS results confirm the fouling release behavior that silicone-based additives may cause. The sharp increase in signal at the serum injection site is due to bulk refractive index change. The amount of non-specific adsorption was estimated from the shift in wavelength from before protein injection to after washing with buffer. The wavelength shifts of the PCB coated and uncoated chips were 0.20 and 2.24nm, respectively, corresponding to 3.4 and 38.1ng/cm, respectively²The serum is adsorbed. The results show that the PCB coated surface has ultra low contamination compared to the uncoated surface under dynamic conditions: (<5.0ng/cm²) Capacity and less than 10% contamination, this is in contrast to MThe icro BCA protein assay kit results were consistent (fig. 24B).

Fibronectin plays a major role in the adhesion of many cell types, including fibroblasts. Thus, the adhesion of NIH3T3 fibroblasts on PCB-coated antifouling surfaces was completely inhibited (fig. 24C). Platelet adhesion is strongly dependent on adsorbed fibrinogen/fibrin, Von Willebrand Factor (VWF), fibronectin, etc., and is 10ng/cm²Fibrinogen adsorption can cause full-scale platelet adhesion (full-scale blood platelet adhesion). The antifouling surface with the PCB hydrophilic hydration layer can effectively protect the surface from contamination and platelet activation (fig. 24D). Although PVC-based materials have shown binding affinity for biofilm-forming bacteria, and gram-positive strains of staphylococcus epidermidis showed slow growth rates with omission, the PCB-coated surface completely inhibited bacterial adhesion (fig. 24E and 24F). Interestingly, bacteria prefer to grow and form biofilms in the valley areas of the textured surface.

Although platelet activation has been considered as a subsequent reaction to adhesion by fibrinogen adsorption, non-platelet/protein-adhesive surfaces do not completely prevent platelet activation. Annexin V is commonly used to detect apoptotic cells by its ability to bind phosphatidylserine, which is a hallmark of platelet apoptosis when it occurs in the outer plasma membrane lobes. Thus, a higher level of annexin V binding indicates more severe apoptosis. The results show that platelets stored in the PCB-coated hydrophilic bag had lower annexin V binding levels compared to those stored in the uncoated bag, indicating that the PCB surface can effectively maintain platelet viability (fig. 25A). Platelets normally remain in a quiescent state, however, when subjected to vascular injury, the shear stress and subsequent cell signaling events due to changes in blood flow velocity can lead to irreversible platelet activation. p-selectin (CD62) is a glycoprotein and is a well-described marker of platelet activation. During platelet activation, p-selectin is transferred from the intracellular granules to the outer membrane. Fig. 25B shows that platelet activation was effectively inhibited on PCB-coated bags due to lower nonspecific platelet-surface interactions compared to the control surface. The level of activation of platelets in the PCB-coated bag at day 8 was comparable to the control sample at day 5. Nevertheless, such platelets still have a very high metabolic activity compared to cryopreserved platelets, and thus the activation level shows a tendency to steadily increase even for platelets in PCB-coated bags. Meanwhile, high metabolic activity and activation of platelets may cause increased glucose consumption (fig. 25G), lactic acid accumulation (fig. 25H), and as a result, if the buffer capacity is exceeded, pH may drop (fig. 25F). A drop in pH to 6.2 or less significantly reduces platelet survival during transfusion. For the PCB coated samples, glucose consumption and lactate accumulation did not seriously affect the pH (>6.8) compared to the control samples. The Morphological Score (MS) was used as a simple indicator of platelet health and was defined as 4 × (dish%) +2 × (sphere%) + (branch%). The higher the percentage of platelets with a healthy disc morphology, the higher the MS value. Even after 8 days, the MS values of platelets stored in the PCB-coated bags showed higher MS (fig. 25C). The binding of VWF to platelets depends on the conformation of the a1 domain that binds to platelet GPIb α, and a higher MFI (mean fluorescence intensity) is associated with a higher binding affinity. This is the criterion for the hemostatic function of platelets. The results show that the MFI of the PCB coated sample at day 8 (320) is higher than the MFI of the control sample at day 5 (300) (fig. 25D). All the platelet evaluation results show that the zwitterionic PCB copolymer surface can effectively relieve platelet damage caused by nonspecific interaction, so that the polymer and surface modification strategies have larger clinical application potential.

In summary, in one aspect, the present invention provides a multifunctional random amphiphilic zwitterionic copolymer composed of super-hydrophilic Carboxybetaine (CB) units, hydrophobic N-Butyl Methacrylate (BMA) units, and photosensitive N- (4-benzoylphenyl) acrylamide units (BPAA), which is used as a surface coating material. The polymer can non-invasively endow the super-hydrophilicity and the antifouling capability to the commercial hydrophobic platelet storage bag through a simple and effective dip coating method. Human platelets stored in this PCB copolymer coated platelet bag show significantly improved properties (e.g., higher viability, lower activation rate, higher morphology score, and higher binding affinity to VWF) compared to current standard storage methods. This coating strategy provides a simple and effective large-scale application via a dip coating process. Thus, the technique is expected to be useful in a wide range of medical and engineering applications, particularly for extending the shelf life of human platelets beyond current standard methods.

Example 4 describes the preparation, characterization and use of representative zwitterionic/hydrophobic/photoreactive copolymers of the present invention.

Zwitterionic copolymer coating composition and substrate coated surface

In another aspect of the present invention, a coating composition is provided. The coating composition includes a zwitterionic copolymer (e.g., a copolymer of formula (I), (II), (III), or (IV)) as described herein. In addition to the zwitterionic copolymer, the coating composition optionally includes a carrier or excipient effective to deliver the zwitterionic copolymer to the surface to be coated. Representative carriers include solvents, such as organic solvents, aqueous solvents, and mixed organic and aqueous solvents, in which the zwitterionic copolymers to be applied are soluble. The coating composition is effective to provide a coating to a surface of a substrate.

For embodiments using zwitterionic copolymers comprising photoreactive groups, the coating is derived from a copolymer as described herein. In the process of fixing the copolymer to the surface, the copolymer coated on the surface is irradiated to achieve copolymer crosslinking, and depending on the surface, the copolymer is crosslinked to the surface. Thus, for these embodiments, the coating comprises a crosslinked zwitterionic copolymer and, depending on the surface, a crosslinked zwitterionic copolymer that is also crosslinked to the surface.

In another aspect, the present invention provides a substrate having at least a portion or all (e.g., externally or internally) of its surface coated with a copolymer of the present invention (e.g., a copolymer of formula (I), (II), (III), or (IV)) or a composition comprising a copolymer of the present invention.

Suitable substrates have a surface that is a hydrocarbon-based surface. Suitable surfaces include plastic surfaces and polymeric surfaces. Representative surfaces include polyolefin, polyester, polycarbonate, Polyurethane (PU), Polysulfone (PSF), poly (ether sulfone) (PES), polyamide, polyacrylic, polyimide, aromatic polyester, Polyethylene (PE), polypropylene (PP), Polystyrene (PS)), poly (ethylene terephthalate) (PET), polyvinyl chloride (PVC), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), and poly (methyl methacrylate) (PMMA) surfaces. In some embodiments, the surface is a polyvinyl chloride surface or a polyurethane surface. In other embodiments, the surface is a cellulose or cellulose acetate surface.

Other suitable surfaces include metal, metal alloy and ceramic surfaces.

In some embodiments, the present invention provides a medical device having at least a portion or all of its surface coated with a copolymer of the present invention or providing a composition comprising a copolymer of the present invention. Suitable medical devices include devices having a surface that is a hydrocarbon-based surface. Representative surfaces include polyolefin, polyester, polycarbonate, Polyurethane (PU), Polysulfone (PSF), poly (ether sulfone) (PES), polyamide, polyacrylic, polyimide, aromatic polyester, Polyethylene (PE), polypropylene (PP), Polystyrene (PS), poly (ethylene terephthalate) (PET), polyvinyl chloride (PVC), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), and poly (methyl methacrylate) (PMMA) surfaces. In some embodiments, the surface is a polyvinyl chloride surface or a polyurethane surface. In other embodiments, the surface is a cellulose or cellulose acetate surface.

Representative instruments include instruments that are plates, discs, tubes, tips, catheters, artificial blood vessels, artificial hearts, or artificial lungs.

In one embodiment, the present invention provides a polyvinyl chloride pipe having at least a portion or all of its inner surface coated with the composition or copolymer of the present invention.

In another embodiment, the invention provides a polyurethane tube having at least a portion or all of its interior surface coated with a composition or copolymer of the invention.

In a further embodiment, the invention provides a polysulfone dialysis membrane having at least a portion or all of its surface coated with a composition or copolymer of the invention.

In another embodiment, the invention provides a hydrocarbon-based film container having at least a portion or all of its interior surface coated with a composition or copolymer of the invention.

In another embodiment, the invention provides a platelet storage bag having at least a portion or all of its interior surface coated with a composition or copolymer of the invention.

Other representative devices in which at least one or more surfaces are advantageously coated with the copolymer of the invention or a composition comprising the copolymer of the invention include implantable/non-implantable medical devices from class I, class II or class III.

As noted above, the zwitterionic copolymers described herein can be used to coat blood-contacting surfaces to impart a variety of advantages to those surfaces. The device comprising the blood contacting surface is a component of a hemodialysis device. Components of a hemodialysis device advantageously treated with the zwitterionic copolymers described herein include dialysis membranes (e.g., blood purification membranes) and dialysis tubing (e.g., polyvinyl chloride and polyurethane tubing). The surface of the membrane advantageously coated with a zwitterionic copolymer comprises cellulose, cellulose acetate, poly (sulfone) (PSF), poly (ether sulfone) (PES), poly (dimethylsiloxane) (PMDS), poly (vinylidene fluoride) (PVDF), poly (lactic acid) (PLA), Polyurethane (PU) and polypropylene (PP).

Methods of using zwitterionic copolymers

In a further aspect, the invention provides methods of using the zwitterionic copolymers of the invention (e.g., copolymers of formula (I), (II), (III), or (IV)) and compositions of the copolymers.

In one embodiment, the present invention provides a method for coating a surface of a substrate comprising contacting the surface of the substrate with a copolymer of the present invention (e.g., a copolymer of formula (I), (II), (III), or (IV)) or a composition comprising a copolymer of the present invention.

In a related embodiment, the present invention provides a method for coating a surface of a substrate that is anti-fouling, the method comprising coating at least a portion of the surface of the substrate with a copolymer of the present invention (e.g., a copolymer of formula (I), (III), or (IV)) or a composition comprising a copolymer of the present invention, and irradiating the surface of the substrate with light effective to crosslink the copolymer on the surface.

In another embodiment, the present invention provides a method of antifouling a substrate surface comprising coating at least a portion of the substrate surface with a copolymer of the present invention (e.g., a copolymer of formula (I), (II), (III), or (IV)) or a composition comprising a copolymer of the present invention.

In a related embodiment, the present invention provides a method of antisoiling a substrate surface comprising coating at least a portion of the substrate surface with a copolymer of the present invention (e.g., a copolymer of formula (I), (III), or (IV)) or a composition comprising a copolymer of the present invention, and irradiating the substrate surface with light effective to crosslink the copolymer on the surface.

In a further embodiment, the present invention provides a method of inhibiting blood protein adsorption on a surface of a substrate comprising coating at least a portion of the surface of the substrate with a copolymer of the present invention (e.g., a copolymer of formula (I), (II), (III), or (IV)) or a composition comprising a copolymer of the present invention.

In a related embodiment, the present invention provides a method of inhibiting adsorption of blood proteins onto a substrate surface comprising coating at least a portion of the substrate surface with a copolymer of the present invention (e.g., a copolymer of formula (I), (III), or (IV)) or a composition comprising a copolymer of the present invention, and irradiating the substrate surface with light effective to crosslink the copolymer on the surface.

In another embodiment, the invention provides a method for coating an interior surface of a platelet storage bag comprising contacting the interior surface of the platelet storage bag with a copolymer of the invention (e.g., a copolymer of formula (I), (III), or (IV)) or a composition comprising a copolymer of the invention and irradiating the surface with light effective to crosslink the copolymer on the contacted interior surface of the platelet storage bag.

In another embodiment, the invention provides a method of coating the interior surface of a polyvinyl chloride tube comprising contacting the interior surface of a platelet storage bag with a copolymer of the invention (e.g., a copolymer of formula (I), (III), or (IV)) or a composition comprising a copolymer of the invention and irradiating the surface with light effective to crosslink the copolymer on the contacted interior surface of the polyvinyl chloride tube.

In other embodiments, the present invention provides methods for inhibiting or preventing the leaching of a plasticizer from a substrate surface. In some of these embodiments, the method comprises:

(a) coating at least a portion of the surface of the substrate with a composition comprising a copolymer to provide a coated surface, the copolymer comprising a first repeat unit and a second repeat unit, wherein each of the first repeat units comprises a pendant zwitterionic group, and wherein each of the second repeat units comprises a pendant photoreactive group effective to crosslink the copolymer on the surface to provide the coated surface; and

(b) the coated surface is irradiated with light effective to crosslink the copolymer on the surface, thereby providing a coated surface effective to inhibit or prevent leaching of the plasticizer from the surface.

In some embodiments, the copolymer further comprises third repeating units, wherein each of the third repeating units comprises a pendant hydrophobic group effective to adsorb the copolymer to a surface. Useful zwitterionic copolymers include those of formulae (I), (III) and (IV).

In some of the above embodiments, contacting the surface with the composition comprises immersing the surface in the copolymer or composition. In still other of these embodiments, contacting the surface with the composition comprises spraying, spin coating, brushing, or rolling the copolymer or composition onto the surface.

In some embodiments of the method of the present invention, contacting the surface with, or coating the surface with, the copolymer or copolymer composition comprises immersing the surface in the copolymer or copolymer composition. In still other of these embodiments, contacting the surface with or coating the surface with the copolymer or copolymer composition comprises spraying, spin coating, brushing, or rolling the copolymer or copolymer composition onto the surface.

As used herein, the term "about" refers to ± 5% of the specified value.

The following examples are provided to illustrate, but not to limit, the present invention.

Examples

Example 1

Preparation, characterization and use of representative zwitterionic/hydrophobic copolymers

In this example, the preparation, characterization and use of representative zwitterionic/hydrophobic copolymers of the invention are described.

Material. Carboxybetaine acrylamide, 1-carboxy-N, N-dimethyl-N- (3 '-acrylamidopropyl) ethylammonium inner salt (CB1) and carboxybetaine methacrylate, 2-carboxy-N, N-dimethyl-N- (2' -methacryloyloxyethyl) ethylammonium inner salt (CB2), respectively, were synthesized according to the previously reported methods. The following materials and reagents were obtained from Sigma-Aldrich (st. louis, MO, USA) and used without any further purification: 2,2' -Azobisisobutyronitrile (AIBN), human plasma fibrinogen (Fg), Human Serum Albumin (HSA), human blood gamma-globulin, Sodium Acetate (SA), N- [ tris (hydroxymethyl) methyl]-3-aminopropanesulfonic acid (TAPS), 1-decanethiol, sodium N-dodecylsulfate (SDS), N-hydroxysuccinimide (NHS), N-ethyl-N' - (3-diethylaminopropyl) carbodiimide hydrochloride (EDC). n-Butyl Methacrylate (BMA) was purchased from Tokyo Chemical Industry Co., Ltd. (Portland, Oregon, USA). Anti-fibrinogen antibodies conjugated to horseradish peroxidase (HRP) were purchased from Novus Biologicals (Littleton, CO, USA). O-phenylenediamine dihydrochloride (OPD) was obtained from Pierce (Rockford, Illinois, USA). Phosphate buffered saline (10 ×, solution), hydrogen peroxide (H)₂O₂30% aqueous solution) and hydrochloric acid (HCl) were obtained from Fisher Scientific Co. (Fair law, NJ). Normal human serum (pooled mixed sex) was purchased from biochied Services (Winchester, VA). Micro BCA protein assayKit and RBS^TM35 concentrates were purchased from Thermo Scientific (Waltham, MA). Multi-well plates with ultra-low attachment surfaces were purchased from Corning Costar Corp. (Corning, NY). Ethanol (200 degrees (proof)) was purchased from Decon Labs (King of Prussia, PA). The water was from a Millipore water purification system and had a minimum resistivity of 18.0M Ω cm. Other organic reagents and solvents are commercially available as ultra-pure grade reagents and are used as received.

Synthesis of polymers. Amphiphilic random copolymer poly (CB1-co-BMA) (PCB1) and poly (CB2-co-BMA) (PCB2) were synthesized by conventional radical polymerization methods using AIBN as an initiator, similar methods have been previously reported (Lin, X.; Konno, T.; Ishira, K., Cell-Membrane-Permeable and Cytocompatible polymeric Nanoprobes joined with Molecular beacons. biomacromolecules 2014,15(1), 150. 157; and Lin, X.; Fukazawa, K.; Ishira, K., Photocontaminated information of DNA binding with a water-soluble Polymer copolymer nanoparticles 2014, 2016, 234. 2016). Briefly, the required amounts of CB monomer, BMA (various molar ratios of CB/BMA: 2/8, 3/7, 4/6, 5/5, 6/4, 8/2) and AIBN were dissolved in ethanol. Transferring the solution toVista^TMThe glass tube reactor was further purged with nitrogen at room temperature for 30 minutes. The polymerization was carried out in a sealed glass tube under a nitrogen atmosphere. After the polymerization, the reaction solution was gently dropped into an ether/chloroform mixed solvent to precipitate a copolymer. The copolymer was filtered and dried under vacuum at room temperature for 24 hours before being collected as a white powder. Residual CB monomer was removed by washing the collected white polymer powder with copious Millipore water. Then, the copolymers were filtered again, frozen with liquid nitrogen and treated with a lyophilizer (Labconco co., ltd., Kansas City, MO) at-80 ℃ for 48 hours to convert them into dry white powders. Use of¹H-NMR (AV-500, Bruker, German) confirmed the chemical structure of the purified copolymer, and the polymer was stored frozen at-20 ℃.

Optimization of coating conditions. Polypropylene (PP) substrates (ePlastic, san diego, CA, USA) were cut into 0.5cm × 0.5cm, ultrasonically cleaned in ethanol for 10 minutes and dried at room temperature. CB copolymers having different amphiphilicities were dissolved in ethanol at a concentration of 0.50 wt%, respectively. Each substrate was immersed in the polymer solution for 10 seconds, and then the solvent was evaporated at room temperature under atmospheric pressure in an ethanol vapor protective environment. All modified PP substrates were soaked in phosphate buffered saline (PBS, 1 ×, pH7.4) at room temperature for 1 hour. The substrate was then rinsed with DI water and vacuum dried at room temperature for 24 hours. To determine the effect of polymer concentration on coating efficiency, clean PP substrates were coated with different concentrations (0.03, 0.06, 0.13, 0.25, 0.5 and 1.00 wt%) of CB copolymer for further testing.

To quickly assess the antifouling capacity of each coating, the adsorbed single protein (fibrinogen) was measured by enzyme-linked immunosorbent assay (ELISA). Briefly, polymer coated PP substrates were pre-wetted overnight in PBS and then dipped into 1.0mg/mL fibrinogen in PBS for 1 hour at 25 ℃. After rinsing with fresh PBS, the PP substrate was soaked in an anti-fibrinogen antibody solution conjugated with HRP for 30 minutes at room temperature. Then, the substrate is rinsed again and brought into contact with OPD/H₂O₂The mixed solution is reacted for 15 minutes; the mixed solution contained 1.0mg/ml OPD and 1000-fold H diluted in citrate buffer (1X, pH 5.0)₂O₂. After quenching the reaction with 1.0N HCl, the absorbance at 492nm of each solution was measured using a microplate reader (BioTek Instruments inc., Winooski, VT).

Surface coating on perforated plates and gold sheets. The 96-well plates made from virgin polystyrene were simply modified with CB copolymer using the dip coating solvent evaporation method described above. Commercial 96-well plates with ultra-low adhesion surfaces were compared to uncoated polystyrene 96-well plates for stain resistance. Gold was made from a BK7 glass slide coated with a first layer of titanium film (-2 nm) and a second layer of gold (-48 nm) using an electron beam evaporator. The tablets were sonicated in each solvent for 5 minutes with acetone, DI water and ethanol. Subsequently, they were treated with a UV/ozone cleaner for 30 minutes,they were then immersed in 0.2Mm 1-decanethiol for 24 hours to form a hydrophobic self-assembled monolayer. Finally, the sheet was dip coated with CB polymer using the same procedure as described above. The thickness of the coated polymer layer was measured under dry conditions using a spectroscopic ellipsometer (a-SE; j.a. woolam co., inc., Tokyo, Japan).

Protein adsorption from single protein solution and 100% human serum. The CB random copolymer coatings were tested synthetically for their antifouling ability against single protein and whole human serum. A single blood protein at a concentration of 10% is often used as a standard to assess biological contamination on various surfaces. Therefore, an assessment of the anti-fouling ability against 100% single blood proteins and 100% human serum is attractive and essential for blood contact devices. Prior to testing for protein adsorption, the coated 96-well plates were pre-wetted with DI water at room temperature. The Micro BCA protein assay kit was used to evaluate protein adsorption to human plasma fibrinogen (Fg), Human Serum Albumin (HSA), human gamma globulin, and human serum. Fg. The concentrations of HSA and gamma-globulin were 3.0, 45 and 16mg/mL, respectively, corresponding to 100% of the concentration in human plasma. Normal human serum (100%, pooled mixed sex) was used as received. Briefly, human protein (in PBS, 1x, pH7.4) or undiluted human serum was incubated in pre-wetted wells at 37 ℃ for 2 hours, then rinsed with fresh PBS (1x, pH 7.4). Adsorbed proteins were desorbed in 1.0ml of a 1.0 wt% sodium n-dodecyl sulfate (SDS) solution. Transfer 150 μ L of supernatant to 96-well plate and mix gently with another 150 μ L of bisquinolinecarboxylic acid (BCA) reagent. After incubation for 2 hours at 37 ℃, two BCA molecules are usually combined with one cuprous ion (Cu)⁺¹) Chelation forms a purple product which is separated from Cu by the protein in an alkaline environment⁺²And (4) reducing. Finally, the absorbance at 562nm was measured using a microplate reader. The absorbance at 562nm is linearly related to the increase in the amount of adsorbed protein.

Cell adhesion. NIH3T3 mouse embryo fibroblasts obtained from American type culture Collection (ATCC, Rockville, Md.) were cultured at 37 ℃ in the presence of 5.0% CO₂In a humid atmosphere of (A) is inoculated in polystyreneAlkene tissue culture dish (phi 10cm, 5.0 × 10)⁴Individual cells/mL) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). The confluent cell cultures were passaged using 0.25% trypsin/EDTA. Sterilized CB polymer solution (0.5 wt%, ethanol) was dropped onto the surface of the tissue culture plate and evaporated in a cell culture hood. The coated surface was then washed with PBS (1x, pH7.4) and pre-wetted with DMEM overnight at room temperature. At 37 ℃ in a medium containing 5.0% CO₂In a humidified atmosphere of (2), NIH3T3 cells were cultured at 5.0X 10⁴The concentration of individual cells/mL was seeded in DMEM supplemented with 10% FBS. After 3 days of culture, the medium was changed and the morphology of the cells on the partially coated surface was observed using a Nikon Eclipse TE2000-U microscope (Nikon Instruments, Melville, NY).

Surface functionalization. Surface functionalization was performed on 96-well polystyrene plates coated with PCB 2-37. The surface modification process using dip coating and solvent evaporation methods has been described in detail above. Carboxyl groups on the polymer surface can be readily activated chemically by EDC/NHS and covalently bonded to amino groups of, for example, proteins, enzymes and aptamers/oligonucleotides. Briefly, first, 0.15mL of freshly prepared DI aqueous solution containing 0.1M NHS and 0.4M EDC was added to a coated 96-well plate at 25 ℃ for 30 minutes to activate the carboxylate groups. Second, the solution of EDC and NHS was removed and the well surface was washed 3 times with 10mM SA buffer (pH 5.0). Third, 0.15mL of Fg solution (1.0mg/mL) in 10mM TAPS (pH 8.2) was added to the activated wells and reacted at 25 ℃ for 30 minutes. Subsequently, the functionalized surface was washed 3 times with BA buffer (10mM boric acid and 300mM sodium chloride, pH 9.0) followed by phosphate buffered saline (PBS, 1 ×, pH7.4) and then evaluated for antibody-antigen specific interactions. Following the protein immobilization process described above, the remaining activated carboxyl groups are also deactivated. Fg functionalized 96-well plates were gently rinsed 3 times with fresh PBS. Then, 0.15mL of anti-fibrinogen antibody conjugated to HRP in PBS was added to each well and held at 25 ℃ for 30 minutes. Thereafter, the wells were washed again and mixed with 0.15mL of OPD/H₂O₂The solution was reacted for a further 15 minutes. Quenching the color development by adding 0.15mL of 1.0N hydrochloric acid solutionAnd (4) reacting. The absorbance at 492nm of each solution was measured using the microplate reader described above. For comparison, 96-well plates coated with unactivated CB polymer but contacted with Fg and 96-well plates with activated CB polymer surface but functionalized with HSA were used as controls.

Statistical analysis. All charts and bar graphs are presented as mean ± Standard Deviation (SD) of three or five replicates as described above. Student's st-test was performed to determine if the observed differences were statistically significant.

Example 2

Preparation, characterization and use of the representative zwitterionic/photoreactive copolymer

In this example, the preparation, characterization and use of representative zwitterionic/photoreactive copolymers of the present invention are described.

Material. Carboxybetaine acrylamide, 1-carboxy-N, N-dimethyl-N- (3' -acrylamidopropyl) ethylammonium inner salt (CBAA) were synthesized according to previously reported methods (Zhang, Z.; Vaisocheroov. h.; Cheng, G.; Yang, W.; Xue, H.; Jiang, J.ang., S., Nonfoulg Behavior of polycarboxybeta-graded Surfaces: Structural and Environmental effects. biomacromolecules 2008,9, 2686-. The following materials and reagents were obtained from Sigma-Aldrich (st. louis, MO) and used without any further purification: 2,2' -Azobisisobutyronitrile (AIBN), 1-decylthiol and sodium n-dodecylsulfate (SDS). Phosphate buffered saline (10 ×, solution) was obtained from Fisher Scientific Co. (fairlaw, NJ). Normal human serum (pooled mixed sex) was purchased from biochied Services (Winchester, VA). Micro BCA protein assay kit and RBS^TM35 concentrates were purchased from Thermo Scientific (Waltham, MA). Ethanol (200 degrees) was purchased from Decon Labs (King of Prussia, PA). The water was obtained from a Millipore water purification system and had a minimum resistivity of 18.0M Ω cm. Other organic reagents and solvents are commercially available as ultra-pure grade reagents and are used as received.

Polymer Synthesis and characterization. Synthesis of photosensitive monomer N- (4-benzoyl phenyl) through reaction of methacryloyl chloride and 4-aminobenzophenone) Acrylamide (BPAA). Amphiphilic PCB copolymers, poly (CBAA-co-BPAA), were synthesized by conventional free radical polymerization methods using AIBN as the thermal initiator (similar methods previously reported: Lin, X.; Fukazawa, K.; Ishihara, K., Photonic Polymer Bearing Group for Surface Modification of biomaterials&Interfaces2015,7, 17489-17498). Briefly, the required amounts of CB monomer and initiator were dissolved in ethanol. The solution was transferred to a Pyrex Vista glass tube reactor and purged with nitrogen further for 30 minutes at room temperature. The polymerization was carried out in a sealed glass tube under a nitrogen atmosphere. After the polymerization, the reaction solution was gently dropped into a poor mixed solvent of ether/chloroform to precipitate the copolymer. The copolymer was filtered and dried under vacuum at room temperature for 24 hours before being collected as a white powder. Residual water soluble monomers were removed by dialysis against DI water for 4 days. The copolymer was then frozen with liquid nitrogen and treated with a lyophilizer (Labconco co., ltd., Kansas City, MO) at-80 ℃ for 48 hours. Use of¹H NMR (AV-500, Bruker, Germany) confirmed the chemical structure of the purified copolymer, and the polymer was stored frozen at-20 ℃. UV light (312nm, 600 mJ/cm)²) Applied to the polymer and evaluated for light sensitivity using a Varian Cary 5000UV-Vis-NIR spectrophotometer.

Surface modification. Medical grade PVC tubing obtained from Streamline air System Set (Medisystems Corporation, MA) was cut into 10cm lengths. The PCB copolymer solution (0.5 wt%, ethanol) was then filled into the tube, which was then sealed and placed on a rotator for 10 minutes to ensure that the entire inner surface was in contact with the polymer solution. Then, the polymer solution was removed and the coated tube was blown with dry air at room temperature. External UV light (312nm, 600 mJ/cm)²) Applied to the coated tube to stabilize the polymer on the inner surface. The unstable polymer was washed away using sterile PBS (1 ×, pH7.4) and the surface pre-wetted, and then a contamination test was performed by adding human serum. The solution was filter sterilized through a 0.45 μm filter.

Surface characterization. The surface wettability of each sample was characterized by measuring the water contact angle. Will distillWater was added to the uncoated and PCB coated PVC tubes and the water contact angle was observed. X-ray photoelectron spectroscopy (XPS) was performed using a Kratos AXIS Ultra DLD spectrometer. Four different samples were analyzed for measured spectra and atoms: (a) PCB copolymer, (b) uncoated commercial PVC pipe, (c) PCB coated commercial PVC pipe stored for 1 week under dry conditions, and (d) PCB coated commercial PVC pipe stored for 3 weeks under dry conditions.

Assessment of biological contamination. The Micro BCA protein assay kit was used to assess human blood protein adsorption of 100% human serum on PCB-coated commercial PVC tubing. Normal human serum (100%, pooled mixed sex) was used as received. Briefly, PCB-coated and uncoated PVC tubes (10cm long) were filled with 100% human serum and incubated at 37 ℃ for 2 hours. The tubes were then rinsed with fresh PBS (1x, pH 7.4). Adsorbed proteins were desorbed in a 1.0 wt% Sodium Dodecyl Sulfate (SDS) solution. Transfer 150 μ L of liquid supernatant to a 96-well plate and mix gently with another 150 μ L of bisquinolinecarboxylic acid (BCA) reagent. After incubation for 2 hours at 37 ℃, there is typically two molecules of BCA with one cuprous ion (Cu)⁺¹) Chelation forms a purple reaction product, cuprous ions are removed from Cu by proteins in an alkaline environment⁺²And (4) reducing. Finally, the absorbance at 562nm was measured using a microplate reader. The absorbance at 562nm is linearly related to the increase in the amount of adsorbed protein.

Example 3

Preparation, characterization and prevention of plasticizer leaching from plastic surfaces Use of

In this example, the preparation, characterization and use of representative zwitterionic/photoreactive copolymers of the present invention to prevent leaching of plasticizers from plastic surfaces is described.

Material. Carboxybetaine acrylamide, 1-carboxy-N, N-dimethyl-N- (3' -acrylamidopropyl) ethylammonium inner salt (CBAA) was synthesized as described in example 2. The following materials and reagents were obtained from Sigma-Aldrich (st. louis, MO, USA) and used without any further purification:2,2' -Azobisisobutyronitrile (AIBN) and 1-decanethiol, sodium n-dodecyl sulfate (SDS), poly (ethylene glycol) methacrylate (PEGMA) (average Mn 360). Nile Blue (NB) acrylamide was purchased from Polysciences, Inc (Warrington, PA, USA). Normal human serum (100%, pooled mixed sex) was purchased from biochied Services (Winchester, VA, USA). The MicroBCA protein detection kit and RBS 35 concentrate were purchased from Thermo Scientific (Waltham, MA, USA). Phosphate buffered saline (10x, solution) was obtained from Fisher Scientific Co. (fairlaw, NJ, USA). Normal human serum (pooled mixed sex) was purchased from biochied Services (Winchester, VA, USA). Micro BCA protein assay kit and RBS^TM35 concentrates were purchased from Thermo Scientific (Waltham, MA, USA). Ethanol (200 degrees) was purchased from Decon Labs (King of Prussia, PA, USA). The water was from a Millipore water purification system and had a minimum resistivity of 18.0M Ω cm. Other organic reagents and solvents are commercially available as ultra-pure grade reagents and are used as received.

Polymer Synthesis and characterization. The photosensitive monomer N- (4-benzoylphenyl) acrylamide (BPAA) was synthesized by the reaction between acryloyl chloride and 4-aminobenzophenone. Amphiphilic PCB copolymer poly (CBAA-co-BPAA) was synthesized by a conventional radical polymerization method using AIBN as a thermal initiator and characterized as described in example 2.

Surface modification. Medical grade PVC tubing obtained from Streamline air System Set (Medisystems Corporation, MA, USA) was cut into 15cm lengths. The tube was then filled with PCB copolymer solution (0.5 wt%), which was then sealed and placed on a rotator for 10 minutes to ensure that the entire inner surface was in contact with the polymer solution. Then, the polymer solution was removed and the coated tube was blown with dry air at room temperature. External UV light (312nm, 600 mJ/cm)²) Applied to the coated tube to stabilize the polymer on the inner surface. The physisorbed polymer was washed away using sterile PBS (1x, pH7.4) and the surface pre-wetted, and human serum was added for subsequent testing. To assess the effect of the PCB polymer on platelet quality, commercially available platelet bags (Teruflex, 150mL, Terumo corp) made of plasticized PVC were coated according to the method described aboveand (i) is (a), Tokyo, Japan). The solution was filter sterilized through a 0.45 μm filter. To evaluate the stability of the coating, a PCB polymer labeled with nile blue (PCB-NB) was coated on the PVC pipe according to the method described above. The tubes were filled with sterile PBS (1x, pH7.4) and incubated at 37 ℃ for the desired period of time. UV/Vis absorption spectra (250-800nm) and fluorescence spectra (excitation: 590nm, 200-900nm) of the solutions were obtained using a UV-Vis-NIR spectrophotometer and a fluorescence spectrophotometer.

The SPR discs were ultrasonically cleaned with acetone, DI water and ethanol for 5 minutes in sequence. Subsequently, they were cleaned using a UV/ozone cleaner for 30 minutes and then immersed in 0.2mM 1-decanethiol for 24 hours to form hydrophobic self-assembled monolayers. Commercial PVC tubing and PCB polymer were dissolved in Tetrahydrofuran (THF) and DI water, respectively. The SPR sheet was spin coated with these two solutions in sequence. UV light (312nm, 600 mJ/cm)²) To the surface of the coated sheet. The thickness of the coated polymer layer was measured under dry conditions with a spectroscopic ellipsometer (α -SE; j.a. woolam co., inc., Tokyo, Japan).

Surface characterization. The surface wettability of each sample was characterized by measuring the water contact angle. Distilled water was added to the uncoated and PCB-coated PVC tubes and then the water contact angle was observed within 10 seconds by photographic image analysis. Five measurements were made for each sample. X-ray photoelectron spectroscopy (XPS) was performed using a Kratos AXIS Ultra DLD spectrometer. Four samples were analyzed for measured spectra and atomic composition: (1) PCB copolymer, (2) uncoated commercial PVC pipe, (3) PCB coated commercial PVC pipe stored for 1 week under dry conditions, and (4) PCB coated commercial PVC pipe stored for 3 weeks under dry conditions. The Binding Energy (BE) was corrected using the peak of C1s at 285eV as a reference. XPS measurement spectra, high resolution spectra of C1s, O1 s and N1s, and atomic composition were obtained for surface analysis.

Assessment of biological contamination. PCB coated commercial PVC tubing was evaluated for human blood protein adsorption to 100% human serum under static and dynamic conditions using a Micro BCA protein assay kit and Surface Plasmon Resonance (SPR), respectively. Briefly, PCB-coated and uncoated PVC tubing (10cm length) was filled with 100% human bloodClear and incubate at 37 ℃ for 2 hours. The tubes were then rinsed with fresh PBS (1x, pH 7.4). Adsorbed proteins were desorbed in a 1.0 wt% Sodium Dodecyl Sulfate (SDS) solution. The liquid supernatant was gently mixed with a bisquinolinecarboxylic acid (BCA) reagent. The absorbance at 562nm was measured using a microplate reader (BioTek Instruments inc., Winooski, VT, USA). The adsorption of human blood proteins under dynamic conditions was calculated using SPR equipped with four independent flow channels, temperature control, intensity stabilizer and peristaltic pump for delivery of liquid samples for real-time monitoring of the interaction between the polymer surface and the protein solution. The coated SPR discs were pre-wetted in PBS (1x, pH 7.4). Protein adsorption behavior on the surface of the PCB-coated SPR slide was monitored by subsequently flowing the following solutions at 25 ℃ at four different flow rates (10, 40, 100 and 200 μ L/min) in sequence: (a) PBS solution (1 ×, pH7.4), 10 min; (b) undiluted normal human serum for 10 min; and (c) PBS solution (1X, pH7.4), 10 minutes. Data at 750nm were collected while flowing different solutions and the amount of fouling was quantitatively assessed by determining the wavelength change caused by protein adsorption on the surface.

Polymer cytotoxicity. The cytotoxicity of PCB polymers was examined according to ISO10993-5 guidelines. Briefly, the fast-growing cell line NIH3T3 mouse embryonic fibroblasts were seeded in 12-well plates to produce sub-confluent cultures within 24 hours. As elution samples, coated and uncoated PVC tubes were cut into 0.2g of the same shape, placed into two separate TCPS wells of a plate containing cell culture medium, and then incubated at 37 ℃ for 24 hours. Blank wells of TCPS plates incubated in normal medium were used as negative controls, while a small piece of latex (excised from a pipette bulb and sterilized in 70% ethanol) was used as a positive control. The extraction medium was transferred from the material sample to one secondary confluent cell well for each of the triplicate samples of each test material. The cultures were incubated at 37 ℃ for a further 48 hours and removed at time points of 24 and 48 hours for microscopic examination. Cells were observed for visible signs of toxicity as indicated by any change in normal morphology compared to negative control cells. Reactivity classes with guidelines set by ISO10993-5The ratings were from 0 to 4. The PCB polymer was dissolved in cell culture medium at various concentrations (0.00125, 0.0025, 0.01, and 0.1mg/mL) and transferred to TCPS wells with NIH3T3 cells and incubated at 37 ℃ for an additional 48 hours. The same examination procedure as described above is performed. In addition, the release of cytosolic enzyme Lactate Dehydrogenase (LDH) corresponding to the cytotoxicity of the synthetic PCB polymer was determined using a lactate dehydrogenase activity assay kit (Sigma-Aldrich, st.

Plasticizer leaching. PCB-coated and uncoated PVC tubes (15cm length) were filled with PBS solution (1X, pH7.4) and incubated at 37 ℃ for 24 hours. The solution in the tube was withdrawn at time points of 4, 12 and 24 hours. UV/Vis absorption spectra (200 and 800nm) were obtained using a UV-Vis-NIR spectrophotometer. The absorption values at 275nm wavelength of the coated and uncoated PVC tubes were compared.

Platelet quality. The effect of PCB polymer on platelet quality was evaluated synthetically using a commercially available flexible platelet bag (Teruflex, 150mL, Terumo Corporation, Tokyo, Japan) made from plasticized PVC. A commercially available platelet bag was coated with PCB polymer using a simple dip coating method similar to the protocol described above. The same volume (30mL) of fresh platelet solution was added to the PCB-coated and uncoated commercial bags, respectively. All experiments were performed on a bench-top machine, with samples stored in an incubator on an orbital shaker (15rpm) (5% CO)₂At 25 ℃ C. Annexin V and p-selectin expression, platelet morphology score, and platelet binding ability to Von Willebrand Factor (VWF) were evaluated.

Complement activation. Complement activation was measured based on sC5b-9 using the corresponding human ELISA kit (Quidel, san Diego, USA). Coated PVC tubes (15cm length) were filled with non-activated human serum and sealed with tape, then incubated at 37 ℃ with gentle rotation. After the desired incubation period (90 min), the incubated sera were removed and diluted with the sample buffer provided by the supplier and applied to 96-well plates pre-coated with antibodies to sC5 b-9. The other steps are performed strictly according to the supplier's protocol. The concentration of sC5b-9 was measured as apparent using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA)Absorption of the color substrate (450 nm). Amphiphilic copolymer poly (PEGMA-co-BPAA) (PPB) was prepared by the same polymerization and purification method as PCB. Commercial PVC tubing coated with PPB was used as a positive control sample.

Statistical analysis. All charts and bar graphs are presented as mean ± Standard Deviation (SD) of three or five replicates as described above. A Student's t-test was performed to determine if the observed differences were statistically significant.

Example 4

Preparation, characterization and use of representative zwitterionic/hydrophobic/photoreactive copolymers

In this example, the preparation, characterization and use of representative zwitterionic/hydrophobic/photoreactive copolymers of the present invention are described.

Polymer Synthesis and characterization. A photosensitive monomer N- (4-benzoylphenyl) acrylamide (BPAA) was synthesized as a covalent bonding group. Amphiphilic PCB copolymers, poly (CBAA-co-BMA-co-BPAA), were synthesized by conventional free radical polymerization methods using AIBN as initiator. Briefly, the required amounts of CB monomer and initiator were dissolved in ethanol. The solution was transferred to a Pyrex Vista glass tube reactor and purged with nitrogen further for 30 minutes at room temperature. The polymerization was carried out in a sealed glass tube under a nitrogen atmosphere. After the polymerization, the reaction solution was gently dropped into an ether/chloroform mixed solvent to precipitate a copolymer. The copolymer was filtered and dried under vacuum at room temperature for 24 hours before being collected as a white powder. Residual water soluble monomers were removed by dialysis against DI water for 4 days. The copolymer was then frozen with liquid nitrogen and treated with a lyophilizer (Labconco co., ltd., Kansas City, MO) at-80 ℃ for 48 hours. Use of¹H NMR (AV-500, Bruker, Germany) confirmed the chemical structure of the purified copolymer, and the polymer was stored frozen at-20 ℃. UV light (312nm, 600 mJ/cm)²) Applied to the polymer and evaluated for light sensitivity using a Varian Cary 5000UV-Vis-NIR spectrophotometer.

Surface modification. A commercially available blood platelet bag (Teruflex, 150mL, Terumo corporation i)on, Tokyo, Japan) was rinsed with 10mL of ethanol for 10 seconds and purged with dry air at room temperature. Then 20mL of aqueous PCB polymer solution (0.5 wt%, DI water) was injected into the bag and the bag was placed on a platelet storage shaker for 10 minutes to ensure that the entire inner surface was in contact with the polymer solution. Subsequently, the polymer solution was removed using a syringe and the coated bag was blown with dry air at room temperature overnight. UV light (312nm, 600 mJ/cm)²) Applied on each side of the coated bag to stabilize the polymer on the surface. The unstable polymer was washed away and the surface pre-wetted using sterile PBS (1x, pH7.4) before the addition of human platelet solution. The solution was filter sterilized through a 0.45 μm filter. A similar dip coating process was used to coat small pieces (1 cm. times.1 cm) of a commercially available platelet bag. SPR sheets were made from BK7 glass slides coated with a first titanium film layer (about 2nm) and a second gold layer (about 48nm) using an electron beam evaporator. The tablets were sonicated in each solvent for 5 minutes with acetone, DI water and ethanol. Subsequently, they were treated with a UV/ozone cleaner for 30 minutes, and then immersed in 0.2mM 1-decanethiol for 24 hours to form hydrophobic self-assembled monolayers. Commercially available platelet bags were cut and dissolved at 0.5 wt% into THF solution. The sheet was spin coated sequentially with a commercially available platelet bag/THF solution and a PCB copolymer/DI aqueous solution. UV light (312nm, 600 mJ/cm)²) To the surface of the coated sheet. The thickness of the coated polymer layer was measured under dry conditions with a spectroscopic ellipsometer (α -SE; j.a. woolam co., inc., Tokyo, Japan).

Surface characterization. The surface wettability of each sample was characterized by measuring the contact angle of water and air with a static contact angle goniometer. Distilled water droplets were deposited on the substrate surface and the water contact angle was measured within 10 seconds by photographic analysis. To measure the air contact angle, all samples were immersed in distilled water for 1.0 hour before measurement. The samples were mounted on custom racks and then immersed in distilled water in glass containers. Air bubbles were introduced through the U-shaped needle under each sample in contact with the measurement surface. For each sample, five different points were measured. XPS analysis was performed on five different samples using a Kratos AXIS Ultra DLD spectrometer: (1) PCB copolymer, (2) platelet bag on the market(3) ethanol rinsed commercial platelet bags, (4) PCB coated commercial platelet bags, and (5) PCB coated commercial platelet bags soaked in buffer. XPS measurement spectra, high resolution spectra of C1s, O1 s and N1s, and atomic composition were recorded for surface analysis. Testing of coated bags for O Using a Portable oxygen Meter₂Permeability.

Human blood protein adsorption. PCB-coated commercially available platelet bags were evaluated for human blood protein adsorption to 100% human serum under static and dynamic conditions using a Micro BCA protein assay kit and Surface Plasmon Resonance (SPR), respectively. Normal human serum (100%, pooled mixed sex) was used as received. Briefly, PCB-coated pieces (1 cm. times.1 cm) of a commercially available platelet bag were incubated with serum at 37 ℃ for 2 hours, then rinsed with fresh PBS (1X, pH 7.4). Adsorbed proteins were desorbed in 1.0ml of a 1.0 wt% sodium n-dodecyl sulfate (SDS) solution. Transfer 150 μ L of liquid supernatant to a 96-well plate and mix gently with another 150 μ L of bisquinolinecarboxylic acid (BCA) reagent. After incubation for 2 hours at 37 ℃, there is typically two molecules of BCA with one cuprous ion (Cu)⁺¹) Chelation forms a purple reaction product, cuprous ions are removed from Cu by proteins in an alkaline environment⁺²And (4) reducing. Finally, the absorbance at 562nm was measured using a microplate reader. The absorbance at 562nm is linearly related to the increase in the amount of adsorbed protein. The adsorption of human blood proteins under dynamic conditions was recorded using SPR equipped with four separate flow channels, temperature control, intensity stabilizer and peristaltic pump for delivering liquid samples for real-time monitoring of the interaction between polymer surface and protein. The coated SPR discs were pre-wetted in PBS (1x, pH 7.4). PBS (1X), H before measurement₂O, HCl (0.1N) and PBS (1X, pH7.4) solution thoroughly washed the place through which the liquid flowed. Protein adsorption behavior on the surface of the PCB-coated SPR slide was monitored by subsequently flowing the following solutions at 25 ℃: (a) PBS solution (1X, pH7.4), 10 min, 40. mu.L/min; (b) undiluted normal human serum, 10 min, 40 μ L/min; and (c) PBS solution (1X, pH7.4), 10 minutes, 40. mu.L/min. The wavelength at 750nm was recorded as the flow of the different solutions. Tong (Chinese character of 'tong')The amount of fouling was quantified by measuring the change in wavelength at 750nm before and after protein adsorption.

Cell adhesion. NIH3T3 mouse embryo fibroblasts obtained from the American type culture Collection (ATCC, Rockville, Md., USA) were cultured at 37 ℃ in the presence of 5.0% CO₂In a humidified atmosphere of (2) was inoculated in a polystyrene tissue culture dish (phi 10cm, 5.0X 10)⁴Individual cells/ml) in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS. The confluent cell cultures were passaged using 0.25% trypsin/EDTA. Sterilized CB polymer solution (0.5 wt%, ethanol) was dropped onto the surface of the tissue culture plate and evaporated in a cell culture hood. The coated surface was then irradiated with UV light (312nm, 600 mJ/cm)²) Next, stabilize and wash with PBS (1X, pH 7.4). At 37 deg.C in the presence of 5.0% CO₂In a humidified atmosphere of (2), NIH3T3 cells were cultured at 5.0X 10⁴The concentration of individual cells/mL was seeded in DMEM supplemented with 10% FBS. After 3 days of culture, the medium was changed and the morphology of the cells on the partially coated surface was observed using a Nikon Eclipse TE2000-U microscope (Nikon Instruments, Melville, NY).

Human platelet adhesion. Fresh human platelets were received from Bloodworks Northwest (Seattle, WA) on the day of donation. 500 μ L of Platelet Rich Plasma (PRP) was inoculated on top of the sample in a 24-well plate and incubated at 37 ℃ for 45 minutes. Then, the sample was rinsed with sterile physiological saline (NS) and fixed using 2.5% glutaraldehyde solution at 25 ℃ for 4 hours. After that, the sample was washed again with sterile physiological saline (NS) and dried at 25 ℃. The surface of the sample was observed using a Nikon Eclipse TE2000-U microscope and a scanning electron microscope (SEM; NE-3200M; SEC; Korea).

Bacterial adhesion test. Gram-positive strains of Staphylococcus epidermidis and gram-negative strains of Pseudomonas aeruginosa were grown in Tryptic Soy Broth (TSB) at 37 ℃ for 12 hours with shaking at 200 rpm. Suspension cultures were diluted and then grown for an additional 2 hours in TSB to reach exponential growth phase. When the second suspension culture reached an optical density of 1.0 at 600nm, the bacteria were centrifuged at 8000rpm and resuspendedFloat in sterile PBS (1X, pH7.4) to a concentration of about 1X 10⁸CFU/mL. Exponentially growing bacteria were then placed on the samples in 24-well plates and incubated at 37 ℃ for 24 hours. Then, the sample was gently washed with sterile PBS to remove non-adherent bacteria, and then stained with 50nM SYTO9 (green fluorescent nucleic acid stain). The results were directly observed using a Nikon Eclipse TE2000-U microscope.

Platelet property assessment. Fresh human platelets were received from Bloodworks Northwest (Seattle, WA) on the day of donation. The custom ficoll procedure was used to dilute the leukapheresis product and platelets were expressed at (2.0-4.3). times.10⁸The concentration of individual cells/mL was transferred to a designated bag (Teruflex, Terumo Corporation, Tokyo, Japan). As reported by Bloodworks NW, all stored platelet collections comply with manufacturer guidelines for platelet concentration, total platelet count, and storage volume. Platelet counts were performed using a hematology analyzer (ABX Diagnostics, Irvine, CA). All experiments were performed on a bench-top machine, with samples stored in an incubator on an orbital shaker (15rpm) (5% CO)₂At 25 ℃ C. The same volume (30mL) of fresh platelet solution was added to PCB-coated and uncoated commercial bags (Teruflex, 150mL, Terumo Corporation, Tokyo, Japan) with stirring at room temperature. Here, the expression of annexin V and p-selectin was evaluated separately; pH, glucose and lactate levels of the platelet solution; platelet morphology score and platelet binding ability to Von Willebrand Factor (VWF).

Annexin V. Platelets were harvested after different incubation periods (1, 6, 24, 48, 120, 144, 168, 192, and 216 hours) and incubated in PBS (1X, pH7.4) at 5.0X 10⁶cells/mL, 300g wash for 8 minutes. After discarding the supernatant, 2.0mL of binding buffer (AnnexinV binding buffer, 10x concentrate, BD pharmingen Becton, Dickinson and Company, NJ) reconstituted platelets and gently mixed. Then, 100. mu.L of the suspension was transferred to a 5.0mL culture solution polystyrene flow cytometry tube (falcon. TM., Thermo Fischer Scientific) using 1.0. mu.L of FITCAnnexinV cell apoptosis detection kit I (BD Biosc)ies, excitation/emission: 496/578 nm). Cells were incubated for 20 min at room temperature in the dark. Samples were analyzed after incubation in a FACScan flow cytometer (Becton-Dickinson, San Jose, Calif.) to detect annexin V binding levels. Platelets stored in uncoated bags were used as control samples.

p-selectin (CD62). Platelets were harvested after the same incubation period as indicated above, and they were incubated at 5.0 × 10⁶The cells/mL were resuspended in an aqueous buffer solution containing PBS (1 ×, pH 7.2), 0.2% fetal bovine serum, and 0.09% sodium azide. Then centrifuged at 300g for 8 min. After discarding the supernatant, platelets were stained with 1.0. mu.L of monoclonal mouse anti-human antibody at a final concentration of 1:100 (FITC mouse anti-human CD62P, excitation/emission: 494/520m) and gently homogenized with a vortex mixer. Cells were then incubated for 30 minutes at room temperature in the dark. After incubation, the samples were analyzed in FACSCAN. Platelets stored in uncoated bags were used as control samples.

Morphology score. The healthiest platelets are in the shape of discs, with a maximum score of 400 (meaning 100% disc-like morphology), but fresh donor platelets typically score around 380. Morphology scores are commonly used to predict platelet health as defined by shape, which is defined as:

m ═ 4 × (dish%) +2 × (sphere%) +1 × (arm%)

MFI for VWF binding affinity. Expression of glycoprotein Ib-alpha (BPIb-alpha) (anti-CD 42b fluorescein; Life Technologies, Carlsbad, Calif.) was used. Binding of VWF to platelets was determined using Alexa Fluor 488-labeled polyclonal anti-VWF antibody. Platelets at room temperature at pH7.4, containing 0.9% NaCl, supplemented with 1.0mM MgSO₄Was incubated for 15 minutes in 10mM HEPES buffer. The mid-fluorescence intensity (MFI) of the anti-VWF signal was collected with the condition of fresh platelets set at 100%.

Glucose and lactate levels. Glucose (mmol/L) and lactate (mmol/L) were measured via electrochemical signals on days 1, 3, 5 and 7 using a blood-gas analyzer (ABL800, Radiometer, Copenhagen, Denmark). Glucose measurement for determining glucose in plasma solutionThe concentration of sugar; this serves as a marker for energy supply. Lactate measurement to determine the concentration of lactate in the plasma solution; this serves as a marker for imbalance between tissue oxygen demand and supply. Here, the potential of the electrode chain is recorded using a voltmeter and correlated to the sample concentration using Nernst's equation. During measurement and calibration, the electrode signals were recorded at 0.982 second intervals. An amperometric method using a membrane selective for different substances (glucose or lactate) was used.

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.