ANTIMICROBIAL COATINGS

Abstract

A coating comprising a metal-organic framework, wherein the metal-organic framework having a zeolitic structure comprising at least one multivalent metal species and at least one organic ligand (such as zeolitic imidazolate framework (ZIF)). Said coating has a topography comprising an array of projections, and each projection having at least one tapered distal end. There is also provided a method of coating substrates with the disclosed coating and use of said coating as a disinfectant, an antiseptic, or an antibiotic. Such use is possible because the tapered distal end of the disclosed zeolitic structure exerting higher pressure on any microbial cell that comes into contact with the disclosed coating, thereby piercing through the cell membrane more easily, causing cell deformation and lysis.

Claims

1.-31. (canceled)

32. A method of killing or inhibiting the growth of a microorganism, said method comprising: contacting said microorganism with a coating wherein the coating comprises a topography having an array of projections formed of a zeolitic imidazolate framework (ZIF), wherein the ZIF comprises at least one multivalent metal species and at least one organic ligand, wherein each projection has at least one tapered distal end, and wherein said projections possess a height greater than or equal to 1 micron.

33. The method of claim 32, wherein said multivalent metal species is tetrahedrally coordinated with said organic ligand.

34. The method of claim 32, wherein said multivalent metal species is a divalent, trivalent or tetravalent metal species or selected from the d-block of the Periodic Table of Elements, or selected from the group consisting of Co, Fe, Cu, Zn and mixtures thereof.

35. The method of claim 32, wherein said organic ligand is an imidazole derivative, an imidazolate or a substituted imidazolate.

36. The method of claim 32, wherein said organic ligand is derived from an imidazole having the following structure: ##STR00004## R1 is C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.3-10 cycloalkyl, or phenyl; R2 is H; and each R3 and R4 are independently H, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.3-10 cycloalkyl, or phenyl; wherein each of R1, R3 and R4 is optionally substituted with halogen, amino, hydroxy, C.sub.1-10 alkyl, oxo, cyano, nitro, C.sub.1-10 haloalkyl, C.sub.1-10 alkoxy and C.sub.1-10 haloalkoxy.

37. The method of claim 32, wherein said organic ligand is derived from the compound: ##STR00005##

38. The method of claim 32, wherein said projections are spaced less than 2 m apart, or possess a width from about 0.1 to 5 m.

39. The method of claim 32, wherein the coating is coupled to a substrate surface.

40. The method of claim 39, wherein metal organic framework is prepared by contacting the substrate surface with a reaction mixture comprising said organic ligand and said multivalent metal species.

41. The method of claim 40, wherein said organic ligand is an imidazole, an imidazole derivative, an imidazolate or a substituted imidazolate.

42. The method of claim 40, wherein said organic ligand is 2-methylimidazole.

43. The method of claim 40, wherein said multivalent metal species is species in Zn.

44. The method of claim 41, wherein said organic ligand (Im) and said multivalent metal species (M) are provided in a molar ratio of Im:M of between 4:1 and 20:1.

45. The method of claim 44, wherein the molar ratio of Im:M is 7:1.

46. The method of claim 32, wherein the ZIF has a ZIF-L crystal structure.

47. The method of claim 32, wherein the microorganism is selected from the group consisting of gram-positive bacteria, gram-negative bacteria and fungi.

48. The method of claim 47, wherein the gram-positive bacteria is Staphylococcus aureus.

49. The method of claim 47, wherein the gram-negative bacteria is Escherichia coli or Pseudomonas auruginos.

50. The method of claim 47, wherein the fungi is Candida albicans, yeast.

51. The method of preparing a disinfectant, antiseptic or an antibiotic comprising coating a surface of a substrate with a coating, comprising a topography having an array of projections formed of a zeolitic imidazolate framework (ZIF), wherein the ZIF comprises at least one multivalent metal species and at least one organic ligand, wherein each projection has at least one tapered distal end, and wherein said projections possess a height greater than or equal to 1 micron.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0101] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0102] FIGS. 1a to 1h show field emission scanning electron micrographic images of glass surfaces coated with 2-methylimidazole (2-Me-Im) and Zn at different 2-Me-Im:Zn ratios, according to Example 1. FIG. 1i shows the x-ray diffraction patterns of ZIF powders produced from 2-Me-Im:Zn ratios of 35:1 and 7:1, according to Example 1. FIG. 1j shows the x-ray diffraction patterns of ZIF powder produced from a 2-Me-Im:Zn ratio of 7:1 (ZIF-L powder), ZIF-L coated glass and uncoated glass, according to Example 1.

[0103] FIG. 2a illustrates the similarity of the nano-crystals of FIG. 1b with a sword shape, while FIG. 2b shows field emission scanning electron micrographs of the top and side views of FIG. 1b.

[0104] FIG. 3 shows field emission scanning electron micrographic images of various surfaces coated with a 2-Me-Im:Zn ratio of 7:1, according to Example 2.

[0105] FIG. 4 illustrates the antimicrobial property of a PMMA surface coated with ZIF-L against E. coli, S. aureus and C. albicans, demonstrated in Example 3.

[0106] FIG. 5a illustrates the antimicrobial property of a glass surface coated with ZIF-L against E. coli in static conditions, demonstrated in Example 3. FIG. 5b illustrates the antimicrobial property of a glass surface coated with ZIF-L against S. aureus in static conditions, demonstrated in Example 3.

[0107] FIG. 6 illustrates the antimicrobial property of a glass surface coated with ZIF-L against E. coli under dynamic condition, demonstrated in Example 3.

[0108] FIG. 7a illustrates the leaching test set-up conducted in Example 4. FIG. 7b shows the leaching results when E. coli was grown in contact with Tryptic Soy broth (TSB) supernatant, ZIF-L supernatant and a known antimicrobial reagent IBN-C8.

[0109] FIG. 8a shows a field emission scanning electron micrographic image of the nano-pillar array structure of the cicada wing, while FIG. 8b shows a field emission scanning electron micrographic image of the nano-sword array structure of the disclosed coating.

EXAMPLES

[0110] Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Example 1

[0111] A zeolitic imidazolate framework (ZIF) was grown directly on normal glass surfaces with 2-methylimidazole (2-Me-Im) as the organic ligand and Zn as the metal.

[0112] The glass surface was placed vertically in a reaction container containing 200.0 ml aqueous solution of 2-Me-Im (0.35 mol/L). 20.0 ml Zn(NO.sub.3).sub.2 aqueous solution (0.5 mol/L at a 2-Me-Im:Zn molar ratio of 7:1) was introduced dropwise into the solution. The mixture was stirred at 25 C. for 3 h to obtain a continuous ZIF-L layer. The ZIF-L coated support was washed with ethanol several times to remove any loose powder on the surface. The washed coated support was then dried in an oven at 60 C.

[0113] Surface morphology of the coated surface was investigated using field emission scanning electron microscopy (SEM, JEOL JSM-7400F, Japan) with samples sputter-coated with a 2- to 3-nm layer of platinum to provide a conductive surface.

[0114] The example was repeated with different ratios of 2-Me-Im to Zn2:1, 35:1 and 70:1.

[0115] ZIF coatings on the glass surfaces were produced and the field emission scanning electron micrographic images of the coated surfaces are shown in FIG. 1.

[0116] FIG. 1a shows the top view of the coated surface at a 2-Me-Im:Zn ratio of 2:1, while FIG. 1e shows the side view of this coated surface.

[0117] FIG. 1b shows the top view of the coated surface at a 2-Me-Im:Zn ratio of 7:1, while FIG. 1f shows the side view of this coated surface.

[0118] FIG. 1c shows the top view of the coated surface at a 2-Me-Im:Zn ratio of 35:1, while FIG. 1g shows the side view of this coated surface.

[0119] FIG. 1d shows the top view of the coated surface at a 2-Me-Im:Zn ratio of 70:1, while FIG. 1h shows the side view of this coated surface.

[0120] As shown in FIGS. 1a to h, different ratios of 2-Me-Im/Zn gave very different coating morphologies on the surface of glass. When the 2-Me-Im/Zn ratio was greater than 35 (FIGS. 1c, 1d, 1g and 1h), a continuous dense layer of ZIF coating was formed and was further confirmed to be of a ZIF-8 structure.

[0121] When the 2-Me-Im/Zn ratio was between 4 and 20, and specifically 7 (FIGS. 1b and 1f), a continuous layer of an array of nano-sword projections was formed and was further confirmed to be of a ZIF-L structure. That is, sword-shaped nano-crystals grew closely on top of the glass surface, with the sharp end of the crystals facing upwards, although their shoulders were randomly directed. This is shown in FIG. 2, which illustrates the top and side views of FIG. 1b.

[0122] Distances between the nano-swords projecting from the glass surface were also irregular, but were all less than 2 m.

[0123] The x-ray diffraction patterns of ZIF powders produced from 2-Me-Im:Zn ratios of 35:1 and 7:1 are shown in FIG. 1i. It can be seen that the different ratios produce diffraction patterns with intensities that differ for similar 28 values.

[0124] The x-ray diffraction patterns of ZIF powder produced from a 2-Me-Im:Zn ratio of 7:1 (ZIF-L powder), ZIF-L coated glass and uncoated glass are shown in FIG. 1j.

Example 2

[0125] The steps in Example 1 were repeated for a 2-Me-Im:Zn ratio of 7:1 (resulting in ZIF-L structures), except the surfaces used in this example were varied. In addition to glass as in Example 1, the surfaces used in this example were poly(methyl methacrylate) (PMMA), silicone, filter paper, metal (copper foil), synthetic cellulose fiber (from a disposable face-mask), Teflon tape and wood.

[0126] It was found that ZIF-L could be successfully grown on all of these surfaces. The ZIF-L coatings on these surfaces have similar nano-sword array structures, although with some variations in the array density.

[0127] This is shown in the series of field emission scanning electron micrograph images in FIG. 3. The inset in each of the series of images in FIG. 3 shows the magnified view of the coated substrates. Specifically, FIG. 3a shows ZIF-L nano-sword arrays on PMMA, FIG. 3b shows ZIF-L nano-sword arrays on silicone, Fig. dc shows ZIF-L nano-sword arrays on glass, FIG. 3d shows ZIF-L nano-sword arrays on filter paper, FIG. 3e shows ZIF-L nano-sword arrays on copper foil, FIG. 3f shows ZIF-L nano-sword arrays on synthetic fiber (from face-mask), FIG. 3g shows ZIF-L nano-sword arrays on Teflon tape and FIG. 3h shows ZIF-L nano-sword arrays on wood.

[0128] Therefore, this example shows that ZIF-L could be successfully grown on surfaces made of different materials, such as metal, plastic, wood and glass.

[0129] In addition, this example shows that ZIF-L could be successfully grown on surfaces having various degrees of hydrophilicity, ranging from cellulose fiber which has the highest hydrophilicity (water contacting angle <10 C.) to Teflon which has the highest hydrophobicity (water contacting angle >170 C.).

[0130] This example also shows that ZIF-L could be successfully grown on surfaces having various degrees of smoothness, ranging from smooth surfaces such as glass and Teflon to rough surfaces such as filter paper and wood.

[0131] Importantly, all these ZIF-L nano-array coated surfaces demonstrated strong bactericidal property. Therefore, these results demonstrate that the ZIF-L nano-sword array coating approach is versatile and this simple coating method can be applied to various supports.

Example 3

[0132] In this example, the bactericidal performance of a ZIF-L coated poly(methyl methacrylate) (PMMA) surface against Gram-negative bacteria Escherichia coli (ATCC 8739), Gram-positive bacteria Staphylococcus aureus (ATCC 6538) and the yeast fungus Candida albicans (ATCC 10231) was tested.

[0133] All bacteria and yeast were stored frozen at 80 C., and were grown overnight at 37 C. in Tryptic Soy broth (TSB) prior to experiments. Yeast was grown overnight at 22 C. in Yeast Mold (YM) broth. Subsamples of these cultures were grown for 3 h further and diluted to give an optical density value of 0.07 at 600 nm, corresponding to approx. 310.sup.8 CFU mL.sup.1 (MacFarland's Standard).

[0134] The steps in Example 1 were repeated, except the surface used in this example was a PMMA surface.

[0135] The antimicrobial property of the coated PMMA surface was evaluated using the JIS Z 2801/ISO 22196 method. Briefly, exponentially growing bacteria with OD600=0.07 was further diluted 100 times and used as test inoculum. A hundred and fifty microliters were inoculated onto each samples and then covered with a 40 mm square of plastic film to ensure close contact between the culture and the coating.

[0136] The samples were placed in 90-mm-diameter petri dishes and incubated at 37 C. After 24 h, both the coated samples and controls and cover films were carefully washed with 14.85 ml of cold TSB to re-suspend the bacteria. A viability count was performed by dilution and plating on growth medium agar plates in duplicate and incubation overnight at 37 C. Since zero cannot be plotted on logarithmic scale, one was added to each count when no colony was observed to allow plotting zero counts.

[0137] The results are shown in the graphs of FIG. 4 and evidence the antimicrobial property of a PMMA surface coated with ZIF-L.

[0138] It was found that the ZIF-L nano-sword array, when a 2-Me-Im:Zn ratio of 7:1 was used, was strongly bactericidal against E. coli with a log reduction of 7 in 24 hours. As shown in FIG. 4a, the CFU graph of the 7:1 ratio which resulted in the ZIF-L nano-sword projections has negligible E. coli CFU readings, while the other CFU graphs of the 2:1 ratio, the 35:1 ratio, the 70:1 ratio and the control PMMA surface with no coating have E. coli CFU readings of 10.sup.7 or more. Furthermore, it can be seen from the CFU graphs of the 35:1 ratio and the 70:1 ratio that the ZIF-8 dense coatings were non-bactericidal.

[0139] FIG. 4b shows the 51-day progression of E. coli growth on the control PMMA surface with no coating compared with the PMMA surface coated with ZIF-L. It can be seen that ZIF-L coated PMMA effectively killed E. coli with a log reduction of more than 7 throughout the 51 days. Further, the negligible E. coli CFU readings throughout all 51 days indicate good stability of the ZIF-L nano-sword coated surface.

[0140] FIG. 4c shows the 24-hour progression of S. aureus growth on the control PMMA surface with no coating compared with the PMMA surface coated with ZIF-L. It can be seen that ZIF-L coated PMMA effectively killed S. aureus with a log reduction of 8, resulting in negligible S. aureus CFU readings after 24 hours.

[0141] FIG. 4d shows the 24-hour progression of C. albicans growth on the control PMMA surface with no coating compared with the PMMA surface coated with ZIF-L. It can be seen that ZIF-L coated PMMA effectively killed C. albicans with a log reduction of 4 after 24 hours.

[0142] Therefore, these results demonstrate that the ZIF-L nano-sword array coating approach is highly bactericidal and can efficiently kill a broad spectrum of bacterial strains.

[0143] This example was repeated again to determine the bactericidal performance of a ZIF-L coated glass surface against Gram-negative bacteria Escherichia coli (ATCC 8739) and Gram-positive bacteria Staphylococcus aureus (ATCC 6538).

[0144] The method used to determine bactericidal kinetics was as follows. Bacteria were grown to log phase in TSB and re-suspended in PBS. After adjusting to OD600=0.07, the re-suspended cells were further diluted 10 times. Then 3 ml of the cell suspension was added to the well of a 6-well plate in triplicates with each well containing a 5.2 cm.sup.2 area ZIF-L coated glass sample or plain glass sample as control. The 6-well plate was incubated at 37 C. under constant shaking of 150 rpm. At each time point (0.5, 1, 3 and 6 h), 100 l of the cell suspensions were removed, rescued by a series of 10-fold dilutions with growth medium, and kept on ice until plating. For plating, 100 l of the diluted samples were spread on growth medium agar plates and colonies were counted after overnight incubation at 37 C.

[0145] The results are shown in the graphs of FIG. 5 and FIG. 6 and evidence the antimicrobial property of a glass surface coated with ZIF-L.

[0146] As shown in FIG. 5a and FIG. 5b, the CFU graphs of the 2-Me-Im:Zn ratio of 7:1 which resulted in the ZIF-L nano-sword projections evidence negligible E. coli and S. aureus CFU readings, respectively, in 24 hours and under static condition when compared to the control glass surface with no coating.

[0147] As shown in FIG. 6, the killing kinetics in terms of CFU evidence negligible E. coli after 24 hours at 37 C. in PBS under dynamic condition (i.e. with constant shaking speed of 150 rpm) for the glass coated with ZIF-L when compared to the control glass surface with no coating.

Example 4

[0148] This example was conducted to prove the hypothesis that ZIF-L nano-sword coated surfaces kill bacteria exclusively via physical interaction.

[0149] A set of control experiments were conducted with ZIF-L and ZIF-8 powders to prove this hypothesis. The minimal inhibitory concentration (MIC) of ZIF-L powders synthesized from a solution with a 2-Me-Im:Zn ratio of 7:1 was tested against E. coli, S. aureus and C. albicans. The same test was conducted for ZIF-8 powders, 2-Me-Im alone and Zn(NO.sub.3).sub.2.4H.sub.2O alone. The MIC values in mg/mL are tabulated below.

TABLE-US-00001 TABLE 1 E. coli S. aureus C. albicans 2-Me-Im 12.5 25 6.2 Zn(NO.sub.3).sub.24H.sub.2O 3.1* >50* 0.8 ZIF-L 2.5 2.5 1.2 ZIF-8 >5.0 >5.0 5.0 *The tested chemical formed precipitate in Tryptic Soy broth (TSB) medium.

[0150] It is therefore evident that ZIF-L has a lower MIC than ZIF-8. Therefore, a lower concentration of ZIF-L is required to inhibit E. coli, S. aureus and C. albicans as compared to ZIF-8.

[0151] The solubilities of ZIF-L in H.sub.2O, phosphate-buffered saline (PBS) and Tryptic Soy broth (TSB) (37 C., 24 hours) are 4.6, 316 and 3.6 ppm of Zn concentration respectively, which are far less than its MIC value (5000 ppm). This indicates that the bactericidal effect came from the ZIF-L coating itself and not the leached Zn. This also indicates that the concentration of Zn that leached from the ZIF-L coating into the various solvents is negligible as compared to the concentration of ZIF-L needed to inhibit E. Coli.

[0152] To further exclude the chemical effect from Zn in the bactericidal performance of the ZIF-L nano-sword array, a Pt-coated nano-sword array was prepared and it demonstrated similar bactericidal property against E. coli. Together with the fact that ZIF-8 dense coating surfaces are non-bactericidal, it was concluded that the bactericidal property of ZIF-L nano-sword coated surfaces relies on physical mechanisms rather than biochemical mechanisms.

[0153] A further Zn leaching test was conducted as follows. 5 mg/ml ZIF-L in TSB was incubated at 37 C. for 24 h. After centrifugation, 100 l of the supernatant was inoculated in a hole of 11 cm.sup.2 carved in agar plates previously seeded with a confluent layer of E. coli (see FIG. 7a). The plates were incubated at 37 C. for 24 h, and the presence or absence of inhibition halos was used to assess potential leaching of zinc ions.

[0154] The test was repeated with TSB supernatant and a known solution of antimicrobial reagent IBN-C8 (structure below) at 64 g/ml.

##STR00003##

[0155] As shown in FIG. 7b, inhibition of E. coli (indicated by the arrow pointing to an inhibition halo around the 11 cm.sup.2 hole) was detected only in the sample with IBN-C8. Inhibition of E. coli was not detected when E. coli cells were grown in contact with ZIF-L supernatant.

INDUSTRIAL APPLICABILITY

[0156] The disclosed coating may be grown or prepared directly on a surface of a substrate to be coated. Substrate that can be coated may be made of a wide variety of materials and may have a wide variety of properties.

[0157] The disclosed method of coating the substrate may be simple, economical and scalable. The disclosed method is a novel approach to grow ZIF nano-arrays on surfaces to confer the surface with superior microbicide activity.

[0158] The antimicrobial effect of the disclosed coating is due to a physical microbicidal mechanism, rather than by biochemical reactions. Advantageously, the development of microbial resistance may be avoided. Further advantageously, the disclosed microbicide surfaces may be clean and safe to the user and require no external microbicidal chemicals.

[0159] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.