The present invention relates to an anti-microbial coating and a method for applying said coating to a surface of a substrate.
The invention has been developed primarily for use in protecting articles from contamination by bacterial and/or fungal infection and will be described hereinafter with reference to this application.
The following discussion of the background to the invention is intended to facilitate an understanding of the invention. However, it should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to below, and anywhere else in the specification, was published, known or part of the common general knowledge in Australia or any other country as at the priority date of any one of the claims of this specification.
Microbe infections remain a significant medical concern, often with life-threatening consequences.1-10 This situation has been exacerbated by the emergence of anti-microbial resistance (AMR), which is a direct result of the on-going misuse and over-prescription of anti-microbial agents.4, 6, 9-12 Indeed, resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Enterobacteriaceae (CRE), which are unresponsive to most conventional antibiotics, have been widely reported.13-16 These factors have contributed to a situation where post-operative infections are rapidly increasing, while the last reliable preventative and therapeutic measures are beginning to fail.13,14, 17 Recent economic projections have estimated that AMR could be responsible for upwards of 10 million deaths per annum by 2050,18 equating to US$100 trillion in healthcare costs and reducing world economic output by around 2-3.5%, if new antibacterial therapies are not developed.18, 19 In tandem, fungal cells have been estimated to infect 1.7 billion people annually, resulting in approximately 1.5 million deaths per annum.20, 21 Unfortunately, deaths due to fungal infections are also increasing, with the mortality rate associated with some species often exceeding 50%, which further increases towards 100% if treatment is delayed.20, 21 Despite these figures, the contribution of fungal infections to the global burden of disease remains unrecognised. In Australia, over a five-year period fungal infections cost an estimated $583 million.22 The median cost for one invasive fungal disease (IFD) is AU$30,957, increasing to AU$80,291 if the patient is admitted to an intensive care unit.23
In response, considerable scientific and medical research has focused on the development of surfaces that are capable of mitigating both bacterial and fungal growth and biofilm formation on surfaces.5, 24-26 Initial research has focussed on the addition of anti-microbial or inhibitory agents to the outer surface of biocompatible materials,27, 28 such as antibiotics,29, 30 and polymers,31, 32 among others.3, 29, 30, 33-40 However, due to several disadvantages, such as patient tissue sensitivity, increasing antibiotic resistance,3 toxicity concerns about nanomaterials,41 and dosage complications,3, 39 additive methodologies have become less viable as a long-term, anti-microbial solution. In response, both scientific and medical research has increasingly been focussed on the development of next-generation therapeutic measures, often with the view to target bacterial physiology previously not exploited in current antibiotic measures.5
Numerous avenues have been explored, including the study of nano-particles and nano-materials, as means for the preparation of new therapeutic measures.40, 42-29 More recently, two-dimensional and low-dimensional materials have received considerable attention as potential anti-microbial agents. These materials have included graphene,50 graphene oxide,50-57 molybdenum disulphide,52, 58 and black phosphorus (BP),59-61 in both their pure and composite forms. Of these low-dimensional materials, BP has emerged as a promising nanomaterial with biomedical applications, including drug delivery, biosensing, bioimaging, and as a promising antibacterial technology.62-65 Studies into the anti-microbial activity of BP, and BP-based composites, have solely focussed on the antibacterial properties of BP that has been liquid exfoliated using solvents. However, the antibacterial properties associated with the liquid exfoliants have not been fully explored to date, and thus the role of these solvent in the noted antibacterial properties cannot be ruled out. Further, solvent suspended black phosphorus cannot be used to impart anti-microbial properties to items.
In relation to the use of black phosphorous as an antibacterial agent Sun et al in Nanoscale, 2018, 10, 12543-12553 demonstrates the viability of BP in solution to act as an anti-bacterial agent. This paper teaches the solution exfoliation of black phosphorous leading to black phosphorous in solution. The solution is then stimulated by irradiation by exposure to 3 minutes of irradiation with near infrared. The requirement for stimulation in this way is undesirable as the irradiation can cause damage to healthy cells as well as the desired pathogen. In addition, the data demonstrates that in order to get above 80% death rate needed a concentration of 640 μg/mL (see
In a similar way Xiong et al in Ecotoxicology and Environmental safety 161 (2018) 507-5014 also reports the solvent exfoliation of BP (using sonication) to produce solutions of BP in water. This study reported (see
Ouyang et al in J. mater Chem B, 2018, 6, 6302-6310 reports the solution exfoliation of BP to produce different solutions of BP in basic N-methyl pyrollidone. As with Sun above the solution is then stimulated by irradiation by exposure to 5 minutes of irradiation with near infrared. The results demonstrated that at 38 mg/mL there was an approximate kill rate of 75%.
Finally, Li et all in ACS Appl. Nano mater. 2019, 2, 1202-1209 teaches the solvent based exfoliation of BP (using sonication) in NMP to form solutions of BP nanosheets in solution. The in-solution sheets were then modified with Titanium complexes and use as anti-bacterial agents. The reported data demonstrates that at a concentration of around 35 μg/ml lead to about 20% inhibition.
Accordingly, whilst BP in solution has been shown to be potentially effective as an anti-bacterial agent it relies on the solvent exfoliation of BP which leads to BP in solution. This is undesirable in some respects as there may be side effects bought about by the solvent and it is unclear whether the solvent is playing any part in the reported activity. In addition, the dosages required were relatively high due to solvent dilution effects.
The present invention seeks to provide an anti-microbial coating and a method for applying said coating to a surface of a substrate, which will overcome or substantially ameliorate at least some of the deficiencies of the prior art, or to at least provide an alternative. In particular we seek to provide a coating whereby the amount of black phosphorous is at lower concentrations than thought previously possible using solution based methods.
According to a first aspect of the present invention, there is provided an article comprising a substrate and an anti-microbial coating, wherein the anti-microbial coating comprises at least one solvent-free black phosphorus flake. The applicants have found that by mechanical exfoliation of BP we can provide concentrations of black phosphorous on a surface significantly lower than that reported for solvent exfoliated systems. Indeed, a skilled worker in the art on reading the studies directed towards solution based black phosphorous as discussed above would not be lead to producing a black phosphorous coating at the levels provided in the present application.
In one embodiment the solvent free black phosphorous flake has been exfoliated. Exfoliation of black phosphorous flakes leads to the formation of either a mono or few layer black phosphorus flake. Black phosphorous flakes of this type are essentially “two-dimensional” as their thicknesses are approaching the atomic scale. In one embodiment the black phosphorous flake has from 1 to 5 layers. In one embodiment the black phosphorous flake has from 1 to 4 layers. In one embodiment the black phosphorous flake has from 1 to 3 layers. In one embodiment the black phosphorous flake is a monolayer black phosphorous flake. In one embodiment the black phosphorous flake has 2 layers. In one embodiment the black phosphorous flake has 3 layers.
Suitably, the at least one black phosphorus flake generates reactive oxygen species (ROS) that are active towards at least some types of micro-organisms.
In one embodiment, the anti-microbial coating prevents the growth of, or kills, micro-organisms selected from the group consisting of bacterial cells and fungal cells or spores.
In one embodiment, the anti-microbial coating has a density that falls within a range of from about 0.1 ng of black phosphorus per μm2 of substrate to about 1.0 ng of black phosphorus per μm2 of substrate.
In one embodiment, the anti-microbial coating has a density that falls within a range of from about 0.2 ng of black phosphorus per μm2 of substrate to about 0.6 ng of black phosphorus per μm2 of substrate
In one embodiment, the anti-microbial coating has a density that falls within a range of from about 0.4 ng of black phosphorus per μm2 of substrate.
In one embodiment, the anti-microbial coating prevents the growth of, or kills, one or more bacterial species selected from the group consisting of: Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA) (resistive species), Salmonella typhimurium, and Bacillus cereus.
In one embodiment, the anti-microbial coating prevents the growth of, or kills, one or more fungal species selected from the group consisting of: Candida albicans, Candida auris, sensitive Cryptococcus neoformans, fluconazole-resistant Cryptococcus neoformans and Ampicillin-resistant Cryptococcus neoformans.
In one embodiment, the anti-microbial coating kills at least about 90% of microorganisms within a period of 120 minutes under ambient conditions, wherein the microorganism is selected from the group consisting of: methicillin-resistant Staphylococcus aureus, or Escherichia coli, or Pseudomonas aeruginosa, or Salmonella typhimurium, or Bacillus cereus, or Candida albicans, or Candida auris, or Sensitive Cryptococcus neoformans, or fluconazole-resistant Cryptococcus neoformans, or Ampicillin-resistant Cryptococcus neoformans.
In one embodiment, the at least one black phosphorus flake has an average thickness that falls within a range of from about 10 nm to about 120 nm
In one embodiment, the at least one black phosphorus flake has an average thickness that falls within a range of from about 15 nm to about 90 nm
In one embodiment, the at least one black phosphorus flake has an average lateral dimension that falls within a range of from about 500 nm to about 5 μm.
Preferably, the at least one black phosphorus flake is produced from a black phosphorus crystal by mechanical exfoliation.
Suitably, the mechanical exfoliation is conducted in a solvent-free environment.
In one embodiment, the at least one black phosphorus flake is physically adsorbed on to a surface of the substrate.
In one embodiment, the at least one black phosphorus flake is deposited onto a surface of the substrate by contacting the substrate surface with an applicator having at least one black phosphorus flake in contact with a surface thereof.
In one embodiment, the reactive oxygen species (ROS) are generated when the black phosphorus flake is exposed to atmospheric oxygen.
In one embodiment, the reactive oxygen species (ROS) are generated under ambient conditions.
Suitably, the reactive oxygen species are selected from the group consisting of a singlet oxygen radical (1O2), a hydroxy radical (OH.), a superoxide radical (O2.−) and hydrogen peroxide (H2O2).
In one embodiment, the reactive oxygen species (ROS) are not substantially cytotoxic towards mammalian cells.
In one embodiment, the article is an implant, a medical instrument, a bioscaffold, or a woven or non-woven textile.
Preferably, the substrate is selected from the group consisting of: metal, an alloy, a polymer, a textile, a glass, a ceramic, or any combination thereof.
Preferably, the metal is selected from the group consisting of titanium, gold, stainless steel, aluminium, copper, or any combination thereof.
In one embodiment, the at least one black phosphorous flake is adhered to the substrate.
According to a second aspect of the present invention, there is provided a method of producing an article having an anti-microbial coating, comprising: providing a substrate; depositing at least one black phosphorus flake onto a surface of the substrate in the absence of solvent.
In one embodiment the solvent free black phosphorous flake has been exfoliated. Exfoliation of black phosphorous flakes leads to the formation of either a mono or few layer black phosphorus flake. Black phosphorous flakes of this type are essentially “two-dimensional” as their thicknesses are approaching the atomic scale. In one embodiment the black phosphorous flake has from 1 to 5 layers. In one embodiment the black phosphorous flake has from 1 to 4 layers. In one embodiment the black phosphorous flake has from 1 to 3 layers. In one embodiment the black phosphorous flake is a monolayer black phosphorous flake. In one embodiment the black phosphorous flake has 2 layers. In one embodiment the black phosphorous flake has 3 layers.
Suitably, the at least one black phosphorus flake generates reactive oxygen species (ROS) that are active towards at least some types of micro-organisms.
In one embodiment, the anti-microbial coating prevents the growth of, or kills, micro-organisms selected from the group consisting of bacterial cells, viruses [TBC] and fungal cells or spores.
In one embodiment, the anti-microbial coating has a density that falls within a range of from about 0.1 ng of black phosphorus per μm2 of substrate to about 1.0 ng of black phosphorus per μm2 of substrate
In one embodiment, the anti-microbial coating has a density that falls within a range of from about 0.2 ng of black phosphorus per μm2 of substrate to about 0.6 ng of black phosphorus per μm2 of substrate
In one embodiment, anti-microbial coating has a density of about 0.4 ng of black phosphorus per μm2 of substrate.
In one embodiment, the anti-microbial coating prevents the growth of, or kills, one or more bacterial species selected from the group consisting of: Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA) (resistive species), Salmonella typhimurium or Bacillus cereus.
In one embodiment, the anti-microbial coating prevents the growth of, or kills, one or more fungal species selected from the group consisting of: Candida albicans, Candida auris, sensitive Cryptococcus neoformans, fluconazole-resistant Cryptococcus neoformans or Ampicillin-resistant Cryptococcus neoformans.
In one embodiment, the anti-microbial coating kills at least about 90% of microorganisms within a period of 120 minutes under ambient conditions, wherein the microorganism is selected from the group consisting of: methicillin-resistant Staphylococcus aureus, or Escherichia coli, or Pseudomonas aeruginosa, or Salmonella typhimurium, or Bacillus cereus, or Candida albicans, or Candida auris, or Sensitive Cryptococcus neoformans, or fluconazole-resistant Cryptococcus neoformans, or Ampicillin-resistant Cryptococcus neoformans.
In one embodiment, the at least one black phosphorus flake has an average thickness that falls within a range of from about 10 nm to about 120 nm
In one embodiment, the at least one black phosphorus flake has an average thickness that falls within a range of from about 15 nm to about 90 nm
In one embodiment, the at least one black phosphorus flake has an average lateral dimension that falls within a range of from about 500 nm to about 5 μm.
In one embodiment, the at least one black phosphorus flake is produced from a black phosphorus crystal by mechanical exfoliation.
Suitably, the mechanical exfoliation is conducted in a solvent-free environment.
In one embodiment, the at least one black phosphorus flake is physically adsorbed on to a surface of the substrate.
In one embodiment, the at least one black phosphorus flake is deposited onto a surface of the substrate by contacting the substrate surface with an applicator having at least one black phosphorus flake in contact with a surface thereof.
In one embodiment, the reactive oxygen species (ROS) are generated when the black phosphorus flake is exposed to atmospheric oxygen.
In one embodiment, the reactive oxygen species (ROS) are generated under ambient conditions.
Suitably, the reactive oxygen species are selected from the group consisting of a singlet oxygen radical (1O2), a hydroxy radical (OH.), a superoxide radical (O2.−) and hydrogen peroxide (H2O2).
In one embodiment, the reactive oxygen species (ROS) are not substantially cytotoxic towards mammalian cells.
In one embodiment, the at least one black phosphorous flake is adhered to the substrate.
Preferably, the substrate is selected from the group consisting of: metal, an alloy, a polymer, a textile, a glass, a ceramic, or any combination thereof.
Preferably, the metal is selected from the group consisting of titanium, gold, stainless steel, aluminium, copper, or any combination thereof.
Preferably, the substrate is produced from a medical grade metal or alloy.
In one embodiment, the substrate is a surgical implant.
In one embodiment, the substrate is a bioscaffold.
In one embodiment, the substrate is a woven or non-woven textile.
According to a third aspect of the present invention, there is provided an article produced according to the method of the second aspect.
According to a fourth aspect of the present invention, there is provided a use of at least one solvent-free black phosphorus flake in the manufacture of an anti-microbial coating for a substrate.
According to a fifth aspect of the present invention, there is provided a solvent-free black phosphorous flake for use in an anti-microbial coating on a substrate.
According to a sixth aspect of the present invention, there is provided a method of producing at least one solvent-free black phosphorus flake with anti-microbial activity, comprising: contacting a black phosphorus crystal with an applicator having an adhesive surface to cause the applicator to adhere to a surface of the black phosphorus crystal; and removing the applicator from the surface of the black phosphorous crystal, thereby mechanically exfoliating at least one black phosphorous flake from the black phosphorous crystal.
According to a seventh aspect of the present invention, there is provided an anti-microbial coating, comprising: at least one black phosphorus flake produced in the absence of solvent.
Suitably, the at least one black phosphorus flake generates reactive oxygen species (ROS) that are active towards at least some types of micro-organisms.
According to an eighth aspect of the present invention, there is provided an article comprising a substrate and an anti-microbial coating according to the seventh aspect, wherein the at least one phosphorus flake is deposited on a surface of the substrate in the absence of solvent.
Other aspects of the invention are also disclosed.
Notwithstanding any other forms which may fall within the scope of the present invention, preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:
It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.
The present invention is predicated on the finding that few-layer BP flakes in their native state, being devoid of a solvent, exhibit broad-spectrum anti-microbial activity towards several types of micro-organisms, including bacteria such as, Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), MRSA, Salmonella typhimurium (S. typhimurium), and Bacillus cereus (B. cereus), as well as a number of fungal cells or spores, including Candida albicans (C. albicans), Candida auris (C. auris) and wild strains of Cryptococcus neoformans (C. neoformans (Sensitive)), fluconazole-resistant C. neoformans (C. neoformans FR), and amphotericin B-resistant C. neoformans (C. neoformans AR) cells.
Microscopic studies were used to assess the underlying anti-microbial activity of these native few-layer BP flakes, revealing that reactive oxide species (ROS) formation was integral to the antibacterial mechanism. The inventors have also surprisingly found that mammalian cells (such as the mouse L929 fibroblasts cell line) are substantially unaffected by the same production of reactive oxide species (ROS), with the cells thriving when exposed to identical BP concentrations. Without wanting to be bound by theory, this disparity between the ability of microorganisms and mammalian cells to deal with ROS likely depends on the fact that mammalian cells have improved mechanisms for sequestering ROS and repairing cellular damage induced by ROS. On the other hand, microorganisms, while having limited ability to cope with oxidative stress, often have a more rapid accumulation of cellular damage and therefore are more vulnerable to oxidative damage.
The utility of BP as a surface coating material was also studied for its effectiveness via surface functionalisation techniques. This study for the first time shows efficacy on a medical grade titanium implant opening a pathway to deploy this self-degradable BP at extremely low concentrations to kill a wide-range of pathogenic micro-organisms including resistant species. These few-layer thick coatings are amongst the thinnest anti-microbial coatings to eliminate the current issue of anti-microbial resistance crisis, where only nano-gram quantities of this material will be required.
What follows is a detailed description of the method required to prepare few-layer black phosphorus flakes for use as an anti-microbial coating on a wide range of suitable articles that find useful application in areas, including but not limited to, the medical device industry and healthcare in general.
Results
2.1 Characterization of Few-Layer Black Phosphorus
Black Phosphorus (BP) is a two-dimensional allotrope of phosphorus, with a characteristic non-planar, ridged lattice. As used herein the terms “few-layer BP” or “black phosphorous flakes” refers to the low-dimensional versions of the material, which consists of several to a few-tens of stacked BP sheets held together by weak van der Waal forces.
A schematic of the materials atomic lattice of BP is shown in
It is important to stress here that the inventors surprisingly found that it was possible to prepare these few-layer BP flakes in the absence of solvent. That is, the few-layer BP flakes were not required to be solvent exfoliated, as is often reported in the literature, but rather mechanically exfoliated in the absence of solvent.
The inventors observed that it was possible to produce solvent-free BP flakes having an average thickness that falls within a broad range of from about 10 nm to about 5 μm.
In various examples, the inventors obtained solvent-free BP flakes with thicknesses of within a range of from about 10 nm to about 120 nm, and some within a range of from about 15 nm to about 90 nm.
The tape comprising the solvent-free BP flakes was then placed against the surface of a substrate of interest, and at least one of the BP flakes is touch transferred onto the desired surface.
In one embodiment, it was possible to deposit these solvent-free BP flakes onto the substrate surface via physical adsorption by relying on the native surface-surface interactions forces, without the need to apply an additional fixative.
In other embodiments, it is possible to secure the solvent-free BP flake(s) to the substrate surface with the aid of an additional fixative (not shown).
Critically, BP produced in this fashion is deposited in a concentration density of from about 0.1 ng of black phosphorus per μm2 of substrate to about 1.0 ng of black phosphorus per μm2 of substrate, more preferably from about 0.2 ng of black phosphorus per μm2 of substrate to about 0.6 ng of black phosphorus per μm2 of substrate, with good results being obtained when the concentration density is around 0.4 ng/μm of substrate.66 This concentration is significantly lower than that used when solvent suspended, or solvent stabilised, BP is analysed. Surprisingly, and despite this low concentration, the BP coating was found to have extremely high anti-microbial properties.
The term “concentration density” or “density” as used herein is used to describe how closely the coating molecules are packed on the surface of the substrate. Accordingly, as used herein the terms means the amount of black phosphorous per unit area (eg., cm2) applied to the substrate.
Analysis of the EDX spectra reveals commensurate data, showing that the flake is predominantly phosphorous, with a presence of oxygen species (see
Raman spectroscopic analysis of the BP flakes following mechanical exfoliation was conducted.
Representative AFM height profiles for the different thicknesses of BP flake obtained by mechanically exfoliation are shown in
2.2. Kinetics of the Anti-Microbial Activity of BP Flakes as a Surface Coating
The anti-microbial effect of the BP flakes was assessed as a function of time using CLSM imaging. Two drug resistant microbes, specifically MRSA and C. neoformans FR, were chosen as representative species to probe the time-correlated anti-microbial behaviour of the BP. Initially, control images were obtained for the untreated cells (see
The relative proportion of viable and non-viable cells (live vs. dead) were then assessed at each time interval via a differential staining technique, where intact and damaged cells (or viable and dead cells respectively) were stained with SYTO® 9 (green colour when assessed via CLSM) and propidium iodide (PI, red colour in when assessed via CLSM), respectively. Specifically, SYTO® 9 fluorescent dye permeates both intact (viable) and damaged cell membranes and binds with nucleic acid.72 PI dyes only permeate through damaged (non-viable) cell membranes and bind in higher affinity with nucleic acids to replace SYTO® 9.72 This allows the visual differentiation between viable and non-viable cells, with the earlier fluorescing green when analysed via CLSM and the latter fluorescing red.
Representative CLSM images for each cell type, following treatment for the indicated time, are shown in
C. neoformans FR
The relative numbers of live and dead cells were then determined and displayed as a percentage in the bar chart presented in
Following exposure to the BP coated surfaces, the proportion of non-viable cells was observed to increase with time. Quantitatively, this equated to 29.5%, 55.7%, 79.5%, and 97.9% and 5.5%, 35.0%, 85.1%, and 99.3% of non-viable cells for the MRSA and C. neoformans FR at time intervals of 30, 60, 90, and 120 min, respectively (see Table 1). Importantly, these values were observed to plateau at 120 min of exposure, with no statistically significant increase in anti-microbial activity noted beyond this time point. It is worthy to note that this result highlights a nominal exposure time of approximately 2 hours to achieve maximum anti-microbial activity of approximately 0.4 ng/μm2 of BP material. Importantly, as briefly discussed above, the degree and rate of anti-microbial effect is significantly greater that that observed in liquid exfoliated BP flakes. While the precise reason for this disparity has not been determined (to date) it is likely that in the absence of solvent stabilisation, the BP flakes readily react with the atmosphere under ambient condition leading to a significant increase in ROS production and a resultantly higher anti-microbial activity.
To further access the antimicrobial effectiveness of the deposited BP nanoflakes, the microbial solutions of MRSA and C. neoformans (FR) were exposed to BP nanoflakes and incubated for 48 hours. The CLSM images of the resulting biofilm and the respective percent viability are shown in
Further, this rapid degradation of solvent-free BP flakes provides a further advantage. As the coating degrades over a relatively short period of time, the risk of microorganisms becoming resistant decreases as a function of reduced exposure, and therefore reduced time for adaptation or selection for resistance. This is a feature not shared by the majority of anti-microbial coating currently used and reported in the art.
2.3 Anti-Microbial Activity of Solvent-Free Black Phosphorus
Following the initial time-lapse investigation of the solvent-free BP flakes against MRSA and C. neoformans FR cells, the anti-microbial activity of the BP flakes was assessed against a broad-spectrum of pathogenic microbial species, including the bacteria, E. coli, P. aeruginosa, MRSA, S. typhimurium, and B. cereus, as well as the fungus C. albicans, C. auris, C. neoformans, C. neoformans FR, and C. neoformans AR using CSLM, following 2 hours of exposure to the material. Here, the relative proportion of live and dead cells was again visualized via fluorescent staining in the CSLM images (see
For the control and treated samples, the relative numbers of live and dead cells were then determined and displayed as a percentage in the bar chart presented in
2.4 Assessing the Anti-Microbial Mechanism of Solvent-Free Black Phosphorus
SEM imaging was employed to visualise the microbial species in their native state and following cellular-nanomaterial interactions. Control SEM micrographs of each microbial species investigated (top row) in the absence of BP adsorbed to a flat silicon surface are presented in
Following treatment with BP (see
Following treatment, the bacterial spores also appear damaged (see insets to respective species). Importantly, this reveals that BP-microbial interactions induce cellular defects, which aide to explain the high-number of inactivated cells quantified via CLSM investigation (see
Without being bound by any one particular theory, the inventors believe that this activity towards at least some types of micro-organisms could be attributed to the ability of BP to interact with atmospheric oxygen under ambient conditions and produce reactive oxygen species (ROS). These ROS can comprise of several radicals such as singlet oxygen radicals (1O2), hydroxy radicals (OH.), superoxide radicals (O2.−) and hydrogen peroxide (H2O2). to name a few.66, 73-75
Escherichia coli
Pseudomonas aeruginosa
Salmonella typhimurium
Bacillus cereus
Candida albicans
Candida auris
Cryptococcus neoformans (Sensitive)
Cryptococcus neoformans FR
Cryptococcus neoformans AR
2.4.1 Computational Investigation of the Mechanism of ROS Generation
In order to gain a better understanding of ROS generation by few-layer BP, quantum chemical (QC) calculations exploring possible mechanisms of superoxide and singlet oxygen formation were performed. Previous studies have shown how superoxide radicals can be generated from pristine BP via a light-activation mechanism.76 However, ROS measurements in this study demonstrate that superoxide radicals and singlet oxygen can be generated from few-layer BP in the absence of light. Optimized geometries and band structures of pristine BP, single-defect BP, and single-defect BP reacted with O2 are shown in
Clearly, the presence of an oxygen vacancy or an oxidized defect widens the band gap of the pristine system and introduces defect states. For a vacancy the gap transitions from being direct to indirect, while for the reacted oxygen system the gap remains direct but shifts to the X-point. For both defect systems, a new spin polarized state is introduced within the band gap region which intersects the Fermi level, indicating a partially filled state. This suggests that the system is reactive and likely to interact with other species if present. The presence of unpaired electrons induces a small magnetic moment on the system, which is localized on the atoms surrounding the defect, and explains the splitting of these bands in the band structures. Structurally, significant differences are observed.
At the oxidized defect site, the distance between adjacent non-bonded P atoms decreases from 3.3 Å to 3.0 Å (
2.5. Cytotoxicity of BP Flakes
To assess the cytotoxicity of the BP flakes, the same concentration of BP flakes on surfaces was tested against L929 mouse fibroblasts following a 48-hour exposure to the material (see
To this end, the inventors believe that the reactive oxygen species (ROS) generated as a result of photo-oxidation of the solvent-free BP flakes are not substantially cytotoxic towards mammalian cells. The hemocompatibility was also tested using ˜900 ng against red blood cells (RBC) with the resulting data shown in
2.6. Assessing the Utility of BP Flakes as an Additive Anti-Microbial in a Practical Application
The previously described experiments were conducted on sterilised glass cover-slips. However, such substrates possess little utility beyond the laboratory. As such, few-layer BP (˜0.4 ng/μm2) was deposited on an industrial relevant substrate, specifically commercial pure grade 2 titanium (ASTM) discs, polyethylene terephthalate (PET) sheets, polydimethylsiloxane (PDMS) sheets and a commercially available fabric bandage. The Ti discs had a nominal diameter of 10 mm, and the PET, PDMS and bandage had a nominal size of 1 cm2. The Ti surfaces are widely used in biomedical implants, both PET and PDMS are medically relevant substrates, and the bandages are used as common wound dressings. BP flakes were then mechanically exfoliated on the different substrates, as previously described, under cleanroom conditions to maintain sterile conditions.
The antimicrobial effect of the few-layer BP coated surfaces was then assessed using CLSM images following a 2-hour exposure of the two previously chosen resistant microbes, specifically MRSA and C. neoformans (FR) (see
Again, control systems were investigated for each as-received surface after 2 hours to assess any inherent antimicrobial activity. The proportion of non-viable cells were commensurate with those observed using the glass substrates for MRSA and C. neoformans (FR), with Ti having 8.9% and 4.2%, PET having 8.9% and 4.2%, PDMS having 8.9% and 4.2%, and the bandage having 8.9% and 4.2% respectively. This shows that the as-received surfaces have no inherent antimicrobial activity.
Importantly, once coated with BP material, the Ti surfaces were found to be highly antimicrobial, with 99.6% and 97.0% of MRSA and C. neoformans (FR) found to be non-viable following 2 hours of surface exposure. Similarly, PDMS had 90.5% of MRSA and 96.9% of C. neoformans (FR) non-viable cells. The bandage was also highly effective with 80.8% of MRSA, and 94.3% of C. neoformans (FR) cells, which were non-viable after 2 hours. This is an important finding, as it indicated that few-layer BP (˜0.4 ng/μm2) could be used as a coating's material to medically relevant substrates to achieve the high antimicrobial activity. For the treated PET surface, the percentage of non-viable cells were 83.0% of MRSA and 76.1% of C. neoformans (FR). This lower percentage can be attributed to the lower amount of mechanically exfoliated BP deposited into the PET surface.
Discussion
Nanotechnology, such as the application of nanoparticles, low-dimensional materials, and surface coatings, have emerged as being applicable as next-generation anti-microbials technologies.5, 24, 25, 76 The rationale behind such studies has been to investigate alternative therapies to conventional treatment methods, such as antibiotics. Very recently, a few studies have noted the antibacterial properties of BP-based solutions59, 61, 77, 78 and its composite materials.60, 61
The culmination of this work suggests that BP in solution in μg/mL concentration regimes can be highly antibacterial;59-61, 77, 78 many of these studies, however, also employed additional UV exposure to increase the inherent bactericidal activity,59 while only exploring the efficacy of the material towards a few bacterial species. This information is summarized in Table 3, along with other commonly investigated antibacterial nanomaterials for comparison to the efficacy of BP elucidated in this study. More importantly, these studies all indicate that the primary antibacterial mechanism is the creation of, and interaction of the microbes with, reactive oxide species (ROS).67, 70, 74, 79
BP-based nanomaterials, such as nanoflakes and composites, are capable of producing ROS. This occurs as the material degrades under ambient conditions in air and in solution.66, 73-75, 80 AFM images of this process are shown in
Specifically, BP-based material will produce significant quantities of .OH, .O2−, and H2O2, where the precise concentration of ROS increases as a function of time and the initial base material concentration.66, 67, 70, 73-75, 79, 80 For microbes, such as bacterial and fungal cells, these ROS are capable of inducing cell lysis, causing cell membrane and intercellular damage.
In general, the anti-microbial activity of BP is thought to occur via two main pathways: 1) membrane disruption, following nanomaterial-microbial interaction, which degrades the cell integrity, or 2) by inducing the production of reactive oxide species (ROS) which initiates cell oxidation, oxidative stress, and ultimately cell lysis.
The results of this study are commensurate with this observation, as the microbial cells show significant membrane damage (see
Previously,73 the production of singlet oxygen species (1O2), as well as hydroxyl (OH.) and superoxide (O2.−) radicals species which are produced by solvent-based mechanically exfoliated BP during degradation of the BP upon exposure to a microbial solution have been measured. As such, these ROS species are undoubtedly produced as the BP materials decompose in the presence of microbial solution, such as that studied here. Both AFM and SEM investigation support these degradative process (see
For the bacterial and fungal species investigated, this anti-microbial action renders most surface attached cells non-viable to a high degree of efficacy; however, some differences in activity are noted between species. The bacteria E. coli, P. aeruginosa, MRSA, S. typhimurium, and the fungi C. albicans, C. auris, C. neoformans (Sensitive), C. neoformans FR, and C. neoformans AR were all at least 90% non-viable on average, with some species showing higher degrees of susceptibility to the action of the nanomaterial. Interestingly, B. cereus cells were found to be only ˜80% non-viable, on average, following 2 hours of treatment, indicating that the cells were more resistant to the anti-microbial action of the BP flakes. The genus Bacillus are known to sporulate in response to environmental stress.82-84 The presence of these spores is clearly seen in both the SEM and CLSM images of B. cereus systems.
The bacterial spores produced during this biological survival mechanism are highly resistant, dormant structures, which enable prolonged survival of these organisms in adverse environments,85, 86 such as that produced by the presence of the BP flakes. In this light, it is not surprising that the B. cereus cells were less susceptible to the antibacterial action of the solvent-free BP flakes; however, the level of inactivation achieved, are still considered high for a bactericidal effect towards spore forming bacteria. This can be further enhanced by using thinner BP layers at slightly higher concentrations which will produce higher number of ROS and possibly overcome resistance.
Mammalian cells, specifically L929 mouse fibroblasts and BJ-5TA human fibroblasts, along with RBCs were found to be unharmed by the presence of BP under the same conditions as the microbial cells. The proliferation rate for the BJ-5TA human fibroblast cells was slower than the control in the presence of BP but cell growth still occurred. This is an important distinction, as it shows that the antimicrobial action of the BP nanoflakes is not toxic towards fibroblast cells in this form and concentrations; however, the material can induce high degrees of microbial cell lysis. The results of these studies are comparatively highlighted in Table 3. Notably, the few-layer BP investigated here is in a purer form than the previously reported for composite-based studies,60, 81 and at a significantly lower concentration, without compromising the antimicrobial efficacy. Importantly, previous studies have shown that bacteria are more sensitive to some anti-microbial agents, such as silver, compared to mammalian cells.87 This means that there is a therapeutic window where an anti-microbial material is effective against pathogens while remaining innocuous towards mammalian cells. It is noteworthy that mammalian cells are known to respond to oxidative stress, such as that imposed by ROS, in a different manner than pathogenic, microorganisms.88 The precise nature of these differences is still unclear, and requires further systematic, in-depth studies, but are at least partly attributable to improved ability to sequester ROS and to repair ROS-related cellular damage.
The general utility of BP as an anti-microbial surface additive was established via functionalizing a medical-grade titanium surface with mechanically exfoliated BP. Critically, the anti-microbial activity of the BP was retained at the titanium interface (see
The mechanically exfoliated BP investigated in this study has several advantages over other nanomaterial based anti-microbials reported within the literature (see Table 3), including: 1) the material displays high levels of anti-microbial efficacy towards both bacterial and fungal species, 2) the concentration required to elicit a high anti-microbial response is significantly lower than used for other nanomaterials, 3) the fabrication method is simple, and requires no other processing or stabilisers to elicit the anti-microbial effects, saving on cost of production. This means that it is readily scalable and represents a simple fabrication procedure, making it an accessible production method worldwide, and 4) any supportive surface could be potentially used with the material, as long as it could be preserved in an inert environment prior to use.
Importantly, the solvent free BP flakes are also known to readily degrade as a function of time when exposed to ambient conditions,47-49, 52, 55-57, 64, meaning that the material is by nature biodegradable. The data obtained in this study highlights the broad-spectrum anti-microbial activity of solvent-free BP flakes, with eight medically relevant pathogenic bacterial and fungal species investigated, including drug-sensitive and drug resistant strains. This highlights the wide-reaching utility of native BP as a biocompatible anti-microbial additive. Together, these data indicate solvent-free BP flakes to be a highly effect anti-microbial, which has scope for use in scientific, medical, and industrial settings.
E. coli, P.
C. albicans,
aeruginosa,
C. auris
typhimurium,
cereus
neoformans
E. coli &
S. aureus
E. coli &
S. aureus
E. coli &
S. aureus
E. coli & B.
subtilis
E. coli
E. coli & S.
aureus
E. coli & S.
aureus
E. coli
E. coli & S.
aureus
E. coli
E. coli
E. coli
S. aureus
P.
aeruginosa
coli),
aureus).
coli), 76.5%
Abbreviations: BP: black phosphorus, NMP: N-methyl-2-pyrrolidone, DMPI: N,N′-dimethylpropyleneurea, PPMS: 4-pyridonemethylstyrene, NPs: nano-particles, NB: Not bactericidal, N/A: Not applicable, NR: Not reported. The summary termed “Mechanically Exfoliated BP” is that of this study. The studies highlighted in respect of AU NPs and Graphene oxide represent work that highlights the controversial nature of a class of anti-microbial nanomaterials.
Applications
Based on these findings, the inventors believe that solvent-free black phosphorus flakes could be applied to a wide range of substrates for use in an equally wide range of applications where such high anti-microbial activity would be beneficial.
For instance, the inventors believe that solvent-free BP flakes could be applied to the surface of a surgical implant or medical instrument using an appropriate means, for example an adhesive.
Preferably, the substrate is selected from the group consisting of: metal, an alloy, a polymer, a textile, a glass, a ceramic, or any combination thereof.
Typically, many surgical implants and medical instruments are manufactured from a medical grade metal or alloy. As already demonstrated above, solvent-free BP flakes can be applied as an anti-microbial coating to the surface of titanium; a metal frequently used for implants and medical instruments.
Similarly, the inventors believe that the surfaces of other metal substrates could be modified with an anti-microbial coating prepared from solvent-free BP flakes. Such metals may include, but are not limited to gold, silver, stainless steel, aluminium, copper, or any combination thereof.
In other embodiments, the substrate to be modified with an anti-microbial coating prepared from solvent-free BP flakes may include a bioscaffold, typically formed from a polymer or composite material.
Similarly, the substrate may also be a woven or non-woven textile such as those used in the manufacture of, for example, bandages, medical garments, bed linen and surgical drapes.
Black phosphorus flakes produced in a solvent-free environment has been explored as a versatile anti-microbial surface coating against both anti-microbial sensitive and resistant bacterial and fungal cells. Importantly, nanogram concentrations of the material were found to be highly anti-microbial towards a broad-spectrum of bacteria and fungi, including E. coli, P. aeruginosa, MRSA, S. typhimurium, and B. cereus, as well as the fungal strains, C. albicans, C. auris and sensitive, fluconazole-resistant, and Amphotericin B-resistant C. neoformans cells.
Further, the solvent-free BP material can be easily deposited onto industrially and medically important substrate surfaces, such as medical grade titanium, via a simple mechanical exfoliation procedure, to impart extremely effective anti-microbial character within 2 hours of microbial exposure.
The study demonstrates BP as a next-generation anti-microbial platform for the treatment of bio-interfacial infections, such as that of wound dressings, as well as for the on-demand sterilisation of applicable surfaces, such as medical devices. More broadly, this study provides a facile methodology for the anti-microbial functionalization of a substrate surface via the deposition of BP-based nanomaterials.
Methods and Materials
Phosphorus Synthesis
BP flakes were produced via mechanical exfoliation from a bulk BP crystal (SmartElements). For microbial testing, the BP flakes were exfoliated directly onto glass bottomed petri-dishes (FluoroDish Cell Culture dishes, Part Number: FD35-100, World Precision Instruments, Sarasota, Fla., U.S.A.).
For SEM imaging, the BP flakes were exfoliated onto plasma-cleaned 300 nm SiO2/Si substrates prior to incubation and SEM preparation. BP surfaces undergo photo-oxidisation in ambient conditions, so the samples were prepared and stored in a nitrogen, enclosed (dark, UV protected)) atmosphere prior to use. BP flakes of uniform thicknesses of 25 to 30 nm were identified via optical contrast microscopy. In the field of electronics, few-layer BP is notoriously known to degrade under ambient conditions.74 As such, the exfoliation process was carried out in UV deficient, dark conditions and the samples were stored in inert nitrogen (N2) atmosphere to preserve the integrity of the material prior to use.67, 68
Bacterial Strains, Growth Conditions, and Sample Preparation
All bacterial strains were obtained from the American Type Culture Collection. Specifically, the stains Escherichia coli DH52, Pseudomonas aeruginosa ATCC27853, Methicillin-resistant Staphylococcus aureus ATCC700699, Salmonella typhimurium ATCC13311, and Bacillus cereus ATCC11778 were investigated in the study. This library of species were chosen as medically relevant pathogenic species which contains representatives of both Gram-negative and Gram-positive bacterium.127
Further, they represent the two main morphologies among bacteria: rod and cocci cells, respectively, for comparison, as well as sporulation in the case of B. cereus. For each experiment, bacteria cultures were grown on Luria-Bertani (LB) agar overnight at 37° C. Bacterial cells were collected from the culture via an inoculation loop and suspended in nutrient broth. These planktonic cell suspensions were grown overnight at 37° C. in 5 mL of Luria-Bertani (LB) broth (B.D., U.S.A.) from loop. The density of the bacterial suspensions was then adjusted to OD600=0.1, after collection on at the logarithmic stage of cell growth.
To obtain a mature biofilm, the planktonic cell suspensions were then added into individual glass-bottom Petri dishes (FluoroDish Cell Culture dishes, Part Number: FD35-100, World Precision Instruments, Sarasota, Fla., U.S.A.) which were either bare or coated in few-layer BP, as indicated. Petri dishes were 35 mm in diameter with a 23 mm well, were comprised of plastic walls with a glass-bottom, and importantly were not pre-coated with any materials.
Fungal Strains, Growth Conditions, and Sample Preparation
Fluconazole- and amphotericin B-resistant Cryptococcus neoformans strains C. neoformans strains were obtained from a fluconazole and amphotericin B resistant isolates derived from strain KN99a which was originally produced by Nielsen et al. (K. Nielsen, G. M. Cox, P. Wang, D. L. Toffaletti, J. R. Perfect and J. Heitman, Infection and immunity, 2003, 71, 4831-4841).
The fungi Candida albicans and Candida auris clinical isolates were obtained from South Australia Pathology Laboratory. Fungal cultures were cultured on yeast extract-peptone-dextrose (YPD) plates for 2 days at 30° C. Fungal suspensions were made in YPD liquid medium with the adjusted OD600=0.1. The incubation procedures on BP surfaces were carried out in a manner similar to that used in respect of the bacterial studies.
SEM Characterization
Scanning electron micrographs were obtained using a field-emission scanning electron microscope (FE-SEM). A FEI Verios Scanning Electron Microscopy (VP, Oberkochen, BW, Germany) at 5 kV was used to image the systems using methods previously described.26, 49, 128
Prior to SEM imaging, all samples were chemically fixed, using 3% glutaraldehyde and 3% formaldehyde in sodium cacodylate buffer pH 7.4 (ProSciTec, QLD, Australia), following with dehydration with series of ethanol concentrations (30%, 50%, 70%, 90%, 100%, 100%). Dehydrated samples were further coated with a thin film of gold prior to imaging.
STEM Characterization
TEM images were obtained with a JEOL 2100F microscope (JOEL, Musashino, Akishima, Tokyo, Japan) equipped with a Gatan Orius SC1000 CCD camera and operated at an acceleration voltage of 80 keV. Images were processed and analysed using Digital Micrograph 2.31.
EELS Characterization
EELS data was collected with a nominal spot size of 1.5 nm and spectrometer entrance aperture of 5 mm with a dispersion of 0.3 eV/ch.
AFM Characterization
AFM images were obtained using both a Cypher ES AFM (Oxford Instrument, Asylum Research, Santa Barbara, Calif., USA) and a JPK nanowizard 4 (JPK BioAFM Business, Am Studio 2D, 12489 Berlin, Germany). All images were obtained under ambient conditions. AC240 cantilevers (Oxford Instrument, Asylum Research, Santa Barbara, Calif., USA, nominal spring constant kc=2 N/m) were used for all measurements. When operated in AC mode imaging forces were minimized via a setpoint ratio (Imaging Amplitude (A)/free amplitude (AO)) of >0.7. The JPK instrument was operated in QI mode. All cantilevers were tuned prior to use using the thermal spectrum method in combination with inverse level sensitivity (InVOLs) as measured by force spectroscopy.
Confocal Imaging and Bacterial Cell Viability Analysis
A combination of confocal laser scanning microscopes (CLSM)—A fluoview FV1200 inverted microscope, Olympus, Tokyo, Japan, and a ZEISS LSM 880 Airyscan upright microscope, Oberkochen, Germany—were used to evaluate the proportions of live and dead cells in each bacteria prior to and following exposure to the polymers within a glass-bottom petri dish (FluoroDish Cell Culture dishes, Part Number: FD35-100, World Precision Instruments, Sarasota, Fla., U.S.A.). Cells were dyed using a LIVE/DEAD® BacLight™ Bacterial Viability Kit (including SYTO® 9 and propidium iodide) (Molecular Probes™, Invitrogen, Grand Island, N.Y., USA). Specifically, SYTO® 9 permeated both intact and damaged cell membranes, binding to nucleic acids and fluorescing green when excited by a 485 nm wavelength laser. Propidium iodide (PI) dominantly enters cells that have undergone membrane damage, which are considered to be non-viable, and binds with higher affinity to nucleic acids than SYTO® 9.
Bacterial suspensions were stained according to the manufacturer's protocol.129 Importantly, discrepancies in viability assessment were avoided by ensuring that no green (485 nm) and red (543 nm) fluorescence overlap was observed during image assessment. Furthermore, photobleaching of the SYTO® 9 dye was avoided by limiting each surface location to a single confocal scan. The live and dead cell ratio was quantified using Cell-C (https://sites.google.com/site/cellcsoftware/) providing a meaningful assessment of the antibacterial activity of the surface.130, 131
Raman Spectroscopy
The Raman spectroscopy characterizations were performed using a Horiba LabRAM HR Evolution Micro-Raman system equipped with a 532 nm laser source (100× objective).
ROS detection: The generation of oxidative species via few-layer BP decomposition under dark condition was determined using a series of dyes. The three oxidative species of interest were OH. radical, O2.— radical and 1O2, and were screened for using horseradish peroxidase (HRP) enzyme (Sigma), xanthine oxidase (Sigma) and methylene blue (MB) (Sigma) respectively. Initially, few-layer BP was exfoliated onto 1 cm2 piece of Si wafer and kept in the dark conditions prior to testing. The HRP solution was prepared by diluting 5 mg of the HRP was diluted into 50 mL of milliQ water. The xanthine oxidase solution was prepared by diluting 5 μM of the oxidase in 50 mL of milliQ water. Methyl blue solution was prepared by diluting 100 mg of the MB into 50 mL of milliQ water, then diluting by a factor of 10. The BP treated Si was covered with 3 mL of the prepared solutions, covered and incubated for 2 hours at room temperature. Bare Si wafter was used as the control. Following the incubation, 1 mL of each solution was run through the UV-visible spectrometer (CARY3500 UV-vis spectrophotometer) over a wavelength range of 200-800 nm. The absorbance peak associated with OH. was 350-450 nm, O2.— was 250-350 nm and 1O2 was 500-700 nm.73
Computational Methods: Quantum chemical calculations involving the few-layer BP surface were performed using density functional theory as implemented in the Vienna ab initio Simulation Package (VASP5.4.4).131, 139. The generalized-gradient (GGA) approximation was employed with the Perdew-Burke-Ernzerhof (PBE) exchange-correlation functional140 and projector augmented wave (PAW) method141 to define the ion-electron interaction. An energy cutoff of 466 eV was applied, with a k-point mesh of 5×5×1 for geometry optimizations and 3×3×1 for ab initio molecular dynamics (AIMD) to sample the Brillouin zone. The van der Waals forces were accounted for by the Grimme DFT-D3 approach.142 In all cases the lattice remained fixed to the previously optimized 2×3 supercell of a single layer of BP, with lattice parameters of a=9.895 Å, b=9.245 Å, and c=20 Å, which included a vacuum region of ˜15 Å to minimize interlayer interactions.143 This system was found to have a bandgap of 0.89 eV, consistent with previous studies. The single-defect BP layer was created by removing a single P atom from the top of the pristine BP. Partially oxidized surfaces were created by changing surface P atoms to O, and optimizing the geometry. For geometry optimization calculations, all atomic positions were relaxed until the total energy of the system was converged to 10-4 eV and the Hellman-Feynman force on each relaxed atom was less than 0.01 eV/A. For the AIMD simulations, two molecules of water and two molecules of O2 were added to each system and all atoms were allowed to relax during the simulation. The simulations were performed for 10 μs at 300 K using a time step of 1 fs.
Mammalian Cell Testing (L929 Mouse Fibroblasts)
The L929 fibroblasts were used to test in vitro cytotoxicity of BP flakes. Cell culture media was DMEM solution supplemented with 10% FBS, and 1% penicillin/streptomycin. The cells were grown to 80% confluence before being collected for experiments.
Prior to the cell experiments, BP flakes were exfoliated onto the well of a sterile tissue culture treated 24-well polystyrene plate. L929 cells were seeded at a concentration of 10,000 cells/cm2 and were incubated at 37° C., 5% CO2 for 48 hours. A sterile well plate with no BP flakes were used as a control. L929 cell viability was quantified by using an MTS assays. For this, 100 μL of CellTiter 96@ AQueous One Solution Proliferation Assay (Promega) was added to each well of the 24-well plates. The plates were incubated at 37° C., in the dark for three hours then analysed using a microplate reader (Spectramax Paradigm) at 490 nm absorbance. The absorbance was normalized to that measured from the control cells.
The cells were also visualized using a confocal laser scanning microscope. The cells were first rinsed with PBS and fixed with formalin 4% solution for 15 mins. They were rinsed twice with PBS to remove excess formalin. The cells were permeabilized and blocked with 0.1% Triton X-100 and 1% bovine serum albumin (BSA), respectively, and were washed three times with PBS. Rhodamine Phalloidin and DAPI (ThermoFisher) were used to stain actin filaments and nucleus, respectively. The samples were washed with PBS and stored with 1 mL of PBS at 4° C. for fluorescent microscopy imaging (Zeiss). DAPI staining was identified as blue when assessed via CLSM and Rhodamine Phalloidin was identified as red when assessed via CLSM.
Mammalian Cell Testing (BJ-5TA human fibroblasts): The BJ-5TA fibroblasts (ATCC-CRL 4001) were used to further test the in vitro cytotoxicity of the BP flakes. Cell culture used was DMEM (ThermoFisher) in a 4:1 ratio with Medium 199 (ThermoFisher), supplemented with hygromycin and 10% foetal bovine serum. Prior to the cell experiment, BP flakes were exfoliated onto half the wells of a sterile tissue cell culture 96-well polystyrene plate, with the remaining bare wells used as controls. BJ-5TA cells were seeded at a concentration of 10,000 cells cm−2 and were incubated at 37° C., 5% CO2 for 48 hours. BJ-5TA cell viability was assessed after 48 hours of exposure using 2 μM Calcein-AM (Cayman Chemicals) and 4 μM Ethidium homodimer (Sigma-Aldrich) added into each well and incubated for 30 min in a 37° C., 5% CO2 incubator. The Calcein-AM is a cell-permeable dye that is hydrolysed by intracellular esterases into green-fluorescent calcein under 494 nm wavelength laser. Ethidium homodimer is a cell-impermeable dye that binds to cellular DNA and fluoresces reddish-orange under 493 nm wavelength laser. The cells were visualized using a high throughput fluorescence microscope (Operetta CLS, PerkinElmer) and were counted using the Harmony software (Operetta CLS, PerkinElmer).
Hemolysis: Fresh blood was collected from a donor of this study and analyzed following the protocol outlined by Li et. al.79 Briefly, the RBCs were separated by centrifuging at 1500 rpm for 15 min then the RBCs were washed with sterile PBS three times. The RBCs were then resuspended in 6 mL of PBS and 1.5 mL of the RBC suspension was exposed to either bare glass cover-slips or BP deposited glass cover-slips (1 cm2). Negative controls were obtained by adding 2 mL of ethanol to the RBC suspensions. After incubating for 2 hours at 37° C., 1 mL of the RBC solution was removed and diluted with 2 mL sterile PBS. The absorbance was measured at a wavelength of 576 nm using a CARY3500 UV-vis spectrophotometer.
Medically Relevant Substrates.
ASTM commercially pure grade 2 titanium rods with nominal diameter of 10 mm were used to manufacture Ti substrates. Firstly, Ti rods were cut into individual discs with an approximate thickness of 5 mm using a Secotom 50 cutting machine (Struers, GmbH, Willich, Germany). Ti discs were then polished with silicon carbide grinding papers with a grit size of P1200 until medical grade smoothness was reached. Each Ti discs was then sterilized via ultrasonication successive washes of MilliQ water and ethanol and then exposed to a UV light source for 30 minutes. Discs were then allowed to dry in a Biosafety Cabinet Class II for 12 hours prior to use. Sylgard™ 184 silicone encapsulant kit (Sigma-Aldrich Australia) was used to cure the PDMS, using a 10:1 ratio of Silicon to a curing agent, and was incubated at 37 C for 24 hours. A 1 cm2 piece was then cut and sterilized via soaking in ethanol and allowed to dry in a Biosafety Cabinet Class II for 30 mins before use.
Commercial grade PET (local source) was obtained, a 1 cm2 area was cut out and sterilized via soaking in ethanol and allowed to dry in a Biosafety Cabinet Class II for 30 mins prior to use. Following sterilization, the surfaces were exposed to 1 mL of microbial solution (prepared using the same procedure above) and incubated for 2 hours under dark conditions. After the incubation period, the surfaces were gently rinsed with sterile PBS and prepared for CLSM images (mentioned above). A commercial-grade bandage (Coverplast standard, BSN medical) was obtained and remained sealed before experimentation. The top layer of the bandage pad was used as the deposition site for the BP flakes. Initially, the 10 uL of the microbial solution was spread onto the corresponding agar plate (LB agar for MRSA and YPD for C. neoformans(FR)) and air-dried for 30 seconds. After the excess liquid dried, the bandage was placed onto the agar and incubated overnight (˜16 hours) at 25° C. The bandage was then removed and placed into a sterile glass-bottom petri dish and prepared for CLSM imaged following the outlined procedure above.
Whenever a range is given in the specification, for example, a temperature range, a time range, or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
The indefinite articles “a” and “an,” as used herein in the specification, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
Spatially relative terms, such as “inner,” “outer,” “beneath,” “below,” “lower,” “above,” “upper,” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the Figures. Spatially relative terms may be intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the Figures.
The term “ambient conditions” is to be understood depending on the context in which it is used. When used in the context of assessing the antimicrobial efficacy of a coating “ambient conditions” refers to standard cell culture conditions of 37° C., 5% CO2 and 95% relative humidity.
While the invention has been described in conjunction with a limited number of embodiments, it will be appreciated by those skilled in the art that many alternatives, modifications and variations in light of the foregoing description are possible. Accordingly, the present invention is intended to embrace all such alternatives, modifications and variations as may fall within the spirit and scope of the invention as disclosed.
Where the terms “comprise”, “comprises”, “comprised” or “comprising” are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof.
Number | Date | Country | Kind |
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2020900142 | Jan 2020 | AU | national |
Filing Document | Filing Date | Country | Kind |
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PCT/AU2021/050032 | 1/20/2021 | WO |