ANTIBACTERIAL ARTICLE COMPRISING A POLYMER MATRIX WITH ALIGNED NANOSCALE FLAKES OR PLATELETS

Abstract

An article having an antibacterial surface having an antibacterial surface having the nano scale flakes or platelets arranged essentially aligned to each other and extending out from said surface with a length in the range of 0.5-30 microns. The antibacterial surface is produced by processing a mixture of a polymer matrix material and a filler material comprising the nanoscale flakes or platelets by pressing the mixture through a die while heated to a temperature above a melting temperature of the polymer matrix material. Hereby, the nano scale flakes or platelets become aligned, with their longitudinal directions being oriented in substantially the same direction. A surface of the processed mixture which is oriented essentially perpendicularly to the longitudinal directions of the nano scale flakes or platelets is then etched or ablated to partly expose the nano scale flakes or platelets, thereby making the surface antibacterial.

Claims

1-17. (canceled)

18. A method for producing an antibacterial surface, comprising the steps: providing a polymer matrix material; providing a filler material comprising nanoscale flakes or platelets; processing a mixture of said polymer matrix material and said filler material by pressing said mixture through a die while heated to a temperature above a melting temperature of said polymer matrix material in such a way that the nanoscale flakes or platelets become aligned, with their longitudinal directions being oriented in substantially the same direction; providing a surface of the processed mixture which is oriented essentially perpendicularly to the longitudinal directions of the nanoscale flakes or platelets; etching or ablating the surface to partly expose the nanoscale flakes or platelets, thereby making the surface antibacterial.

19. The method of claim 18, wherein the processing of the mixture is at least one of an extrusion process and an injection molding process.

20. The method of claim 18, wherein the filler material comprises essentially only nanoscale flakes or platelets, and preferably consists of nanoscale flakes or platelets.

21. The method of claim 20, wherein the filler material comprises at least 90 wt-% carbon, and preferably at least 95 wt-%, and more preferably at least 99 wt-%, and most preferably at least 99.9 wt-%.

22. The method of claim 18, wherein the mixture comprises an amount of filler material in the range of 3-40 wt-%, and preferably in the range of 5-30 wt-%, and more preferably in the range of 6-25 wt-%, and more preferably in the range of 7.5-20 wt-%, and most preferably in the range of 10-15 wt-%.

23. The method of claim 18, wherein the nanoscale flakes or platelets are of graphene or graphite.

24. The method of claim 18, wherein the processing provides a homogenous mixture of polymer matrix material and filler material.

25. The method of claim 18, wherein the polymer matrix material is a thermoplastic polymer, and preferably a polymer selected from a group consisting of: polycarbonates (PC), polyvinyl chloride (PVC), polyurethanes, copolymers comprising ethylene, such as polyethylene co-acetate, and polyolefin based elastomers, such as polypropylene (PP), styrene-ethylene-butylene-styrene (SEBS) and polyethylene (PE).

26. The method of claim 18, wherein the step of providing a surface of the processed mixture comprising cutting of the processed material, and preferably in an essentially transversal or longitudinal direction in relation to a flow direction through the die.

27. The method of claim 18, wherein the nanoscale flakes or platelets have an average length in the range of 5-50 microns, and preferably in the range of 10-40 microns, and more preferably in the range of 15-35 microns, and most preferably in the range of 20-30 microns.

28. The method of claim 18, wherein the nanoscale flakes or platelets have an average thickness in the range of 0.01-20 nm, and preferably in the range of 0.1-15 nm, and more preferably in the range of 0.5-10 nm, and most preferably in the range of 1-5 nm.

29. The method of claim 18, wherein the nanoscale flakes or platelets have an average width in the range of 1-30 microns, and preferably in the range of 1-20 microns, and more preferably in the range of 2-15 microns, and most preferably in the range of 3-15 microns.

30. The method of claim 18, wherein the etching or ablating of the surface is made so that the nanoscale flakes or platelets, on an average, extends from the polymer matrix material of the surface by a length in the range of 0.5-30 microns, and preferably in the range of 1-25 microns, and more preferably in the range of 2-20 microns, and most preferably in the range of 3-15 microns.

31. The method of claim 18, wherein the distance between any adjacent nanoscale flakes or platelets in the plane of the surface is less than 10 μm, and preferably less than 5 μm, and most preferably less than 1 μm.

32. An article having an antibacterial surface, wherein the article comprises a substrate comprising a polymer matrix and a filler material comprising nanoscale flakes or platelets, wherein said article comprises an antibacterial surface having the nanoscale flakes or platelets arranged essentially aligned to each other and extending out from said surface with a length in the range of 0.5-30 microns.

33. The article of claim 32, wherein the nanoscale flakes or platelets extend out from the surface by a length in the range of 1-25 microns, and more preferably in the range of 2-20 microns, and most preferably in the range of 3-15 microns.

34. The article of claim 32, wherein the article is provided as a coating, and preferably as a coating on a medical device, such as a catheter, and preferably a urinary catheter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] By way of example embodiments of the invention will now be described with reference to the accompanying drawings in which:

[0058] FIGS. 1a and 1b are flow charts schematically illustrating exemplary embodiments of a production of an antibacterial surface, in accordance with embodiments of the present invention;

[0059] FIGS. 2a and 2b are cross-sectional views in a plane parallel to an extrusion direction, schematically illustrating orientation and alignment of nanoscale flakes or platelets during extrusion in the methods of FIGS. 1a and 1b;

[0060] FIGS. 3a and 3b are cross-sectional views in a plane perpendicular to an extrusion direction, schematically illustrating orientation and alignment of nanoscale flakes or platelets during extrusion in the methods of FIGS. 1a and 1b;

[0061] FIGS. 4a and 4b are cross-sectional views schematically illustrating etching/ablating of a surface to expose the flakes/platelets in the methods of FIGS. 1a and 1b;

[0062] FIG. 5 is a schematic illustration of an extrusion apparatus useable in the methods of FIGS. 1a and 1b; FIGS. 6-8 are schematic illustrations of how orientation and alignment of the flakes/platelets is obtained in methods according to the present invention;

[0063] FIG. 9 are SEM pictures illustrating different antibacterial surfaces in accordance with embodiments of the present invention;

[0064] FIG. 10 are diagram showing the experimentally determined antibacterial activity of the surfaces of FIG. 9;

[0065] FIG. 11 are fluorescence microscope pictures illustrating live/dead bacterial viability of the surfaces of FIG. 9;

[0066] FIG. 12 are SEM pictures showing bacteria in contact with the surfaces of FIG. 9; and

[0067] FIG. 13 is a schematic illustration of the antibacterial activity of antibacterial surfaces in accordance with embodiments of the present invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0068] In the following detailed description preferred embodiments of the invention will be described. However, it is to be understood that features of the different embodiments are exchangeable between the embodiments and may be combined in different ways, unless anything else is specifically indicated. It may also be noted that, for the sake of clarity, the dimensions of certain components illustrated in the drawings may differ from the corresponding dimensions in real-life implementations of the invention.

[0069] In a production method in accordance with a first embodiment, as illustrated schematically in the flow chart of FIG. 1a, the method comprising mixing of a polymer matrix material and a filler material, wherein the filler material comprises nanoscale flakes or platelets, step S1. Thereafter, in step S2, the mixture is processed to align the platelets within the mixture. This can e.g. be made by extrusion of the mixture, but other types of processing, such as injection molding, can alternatively be used.

[0070] Then, in a step S3, a surface is formed in the extruded part, so that the surface extends essentially perpendicularly to the longitudinal direction of the aligned platelets. This forming may e.g. be made by cutting the extruded part in a certain direction, such as in a transversal or longitudinal direction in relation to the extrusion direction.

[0071] Finally, in step S4, the surface is etched or ablated, thereby removing the uppermost part of the polymer matrix material, so that the platelets previously being embedded close to the surface now extend out from the surface.

[0072] As discussed in the foregoing, different polymer matrix materials and filler materials may be used. In a specific embodiment, LDPE is used as a polymer matrix material, and GNP is used as filler material.

[0073] A method for producing an antibacterial surface using LDPE and GNP is illustrated in FIG. 1b.

[0074] Here, in step S1a, the GNP is initially mixed in a liquid, such as acetone, to deagglomerate the platelets from each other. However, depending on the quality etc of the filler material, such initial deagglomeration may be unnecessary, and may then be omitted.

[0075] In step S1b, the GNP and acetone suspension may be homogenized and further deagglomerated in an additional mixing step. However, this step is also optional, and may in many cases be omitted.

[0076] Exfoliation, e.g. in a high shear mixer, may also be used in order to reduce the thickness of the nanoplatelets by separation of their layers.

[0077] The suspension is then, in step S1c, mixed with LDPE, preferably provided in the form of powder. The LDPE and GNP are hereby preferably mixed and stirred to form a homogeneous mixture, and until the acetone has evaporated. However, other ways of forming the mixture are feasible, and in certain embodiments, mixing and deagglomeration can be made directly in the subsequent processing, such as in an extrusion process.

[0078] The mixture is then, step S2a, feed into an extruder, whereby the platelets of the filler material will be aligned so that their longitudinal directions extend generally in the extrusion direction. Depending on the materials used, how much homogenization and deagglomeration that was made prior to extrusion, how much alignment that is required, etc, it may be necessary to run the material through the extruder more than one time, such as 2-10 times, and in particular 2-5 times. As a rule of thumb, the platelets will be more aligned and better dispersed with every additional round of extrusion.

[0079] When a sufficient degree of deagglomeration and alignment has been obtained, step S2b, which may be determined by evaluation of the produced material, or based on empirical knowledge from previous batches, the extruded material is removed. The material is then, in step S3′, cut in a direction perpendicular to the length direction of the aligned platelets within the material. The cutting may e.g. occur longitudinally or transversally, as seen in the extrusion direction.

[0080] Finally, in step S4, the surface is etched/ablated, thereby to expose the outermost platelets, but while still being partly embedded within the polymer matrix material.

[0081] The process is further illustrated schematically in FIGS. 2-4.

[0082] In FIG. 2a, a schematic cross-sectional view in the longitudinal direction of the material 1 is illustrated. Prior to extrusion, or after an insufficient number of extrusion rounds/loops, the platelets 3 are embedded in the polymer matrix material 2, but arranged in random, non-aligned orientations. After sufficient number of rounds in the extruder, such as 1-3 rounds, the platelets will be aligned in the extrusion direction, as schematically illustrated in FIG. 2b.

[0083] This material may then be cut in a transversal direction, illustrated with dashed line 4, to form the antibacterial surface.

[0084] In addition to being aligned in their length directions, the platelets will also be aligned in their width directions. This alignment occurs around concentric circles around the center of the extruded part, so that each major side of the platelets is facing either towards the center or away from the center. This is schematically illustrated in FIGS. 3a and 3b.

[0085] In FIG. 3a, a schematic cross-sectional view in the circumferential direction of the material 1 is illustrated. Prior to extrusion, or after an insufficient number of extrusion rounds, the platelets 3 are embedded in the polymer matrix material 2, but arranged in random, non-aligned orientations. After sufficient number of rounds in the extruder, such as 1-3 rounds, the platelets will be aligned in concentric circles around the center, as schematically illustrated in FIG. 3b.

[0086] This material may then be cut in a longitudinal direction, illustrated with dashed line 4, to form the antibacterial surface.

[0087] The surface is etched/ablated to remove loose platelets, and to lower the surface of the polymer matrix material. This is illustrated schematically in FIGS. 4a and 4b, where FIG. 4a illustrate the material prior to etching/ablating, and where FIG. 4b illustrate the same material after etching/ablating. After etching/ablating, the platelets 3 will extend out from the surface and from the polymer matrix material, but still be embedded in the polymer matrix material at the other ends.

[0088] The removal of the polymer matrix material at the surface can be obtained by etching, such as chemical wet etching, e.g. by use of strong acids. However, other methods may also be used, such as plasma etching/ablation, laser etching/ablation, etc.

[0089] By the above-discussed processing, the platelets are orientated in the desired way within the produced material, and are further sufficiently deagglomerated and dispersed.

[0090] Extrusion processing is per se well known, and need not be discussed in detail. An exemplary extruder is illustrated in FIG. 5. The extruder 10 may have an inlet, a so-called hopper 11, an extruder screw 12, one or several heaters 13, which may provide zones T1-T4 with controllable temperatures, and a nozzle or die 14.

[0091] The polymer matrix material is heated to a temperature above the melting temperature during extrusion, and the velocity gradient inside the die will form a parabolic velocity profile, as illustrated in FIGS. 6 and 7. Deviations from this will occur if the local shear rates fall within the shear-thinning region of the viscosity function. Therein, the nanoplatelets will align with the principal axis in the extrusion flow direction and the secondary axis along concentric iso-velocity cylindrical planes, as illustrated in FIG. 6, schematically illustrating the flow induced morphology. Deviations from the expected orientation could occur in the presence of low velocity gradients, e.g. in the center of the flow channel especially for non-Newtonian fluids where the velocity profile is flattened in the center.

[0092] In addition to the velocity gradient requirements for orientation, the induced stresses—shear stresses in the case of steady state flow inside the die—should preferably be sufficiently high to ensure a good breaking of filler agglomerates.

[0093] It has been shown experimentally given the same kinematic conditions, a polymer matrix material with higher viscosity resulted in the most consistent de-agglomeration and subsequent orientation and was selected for the study. FIG. 8 presents the viscosity function and flow curve for LDPE. The screw speed was thus chosen such that the apparent shear rate (V) inside the die would fall as close as possible to the Newtonian plateau of the polymer matrix. Numerical solution of the Poiseuille flow inside the die using the viscosity function in FIG. 8 for a screw speed of 30 rpm is shown in FIG. 7.

[0094] Generally, it is preferred to use a polymer matrix material having relatively high viscosity, since it facilitates the process of orienting and aligning the flakes/platelets. However, materials having lower viscosity may also be used, and then preferably with use of higher pressure in the extrusion process.

[0095] Generally, the shear stress is a function of viscosity and shear rate, and it is believed that a high shear stress is beneficial, since it improves de-agglomeration of the flakes/platelets, and that a high shear rate is beneficial, since it improves orientation and alignment of the flakes/platelets.

[0096] Generally, it is also preferred to provide the processing, such as the extrusion, at relatively low temperatures, above but relatively close to the melting temperature of the polymer matrix material. Preferably, the processing temperature(s) is/are less than 100 degrees above the melting temperature of the polymer matrix material, and preferably less than 50 degrees above said melting temperature.

[0097] The die may have any shape. In one embodiment, the die has a circular opening, producing an extrudate in the form of a cylindrical rod. However, other shapes, are also feasible, such as using a rectangular die opening, to produce an extrudate in the form of a plate or the like.

[0098] In some applications, the extrusion or injection molding can be used to form a device, such as a medical device, whereby the antibacterial surface may be provided directly on a surface of the produced device. However, alternatively, the antibacterial surface can be produced separately, such as in the form of sliced stripes, ribbons or the like, which may then be attached to a surface of a device like a coating.

[0099] In case the antibacterial surface is produced in the forms of stripes or ribbons, the rest of the material may be recycled into the process, and again used for extrusion or injection molding.

Examples and Experiments

[0100] A practical example of preparation of GNP-LDPE nanocomposites will now be discussed.

[0101] The GNP-LDPE composites were obtained by the method illustrated in FIG. 1b. For this purpose, low density polyethylene (Borealis AB, Stenungsund, Sweden) was used. Characteristics of the LDPE, based on Gel Permeation Chromatography and Differential Scanning calorimetry is presented in Table 1 below.

[0102] GNP grade M25 was obtained from XG Sciences (Lansing, USA) for use as a filler. The properties of GNP as stated by the manufacturer are presented in Table 1 below, extracted from product data sheet.

TABLE-US-00001 TABLE 1 Materials parameters GNP LDPE d.sub.avg [μM] 25 M.sub.w [kg/mol] 92 A [m.sup.2] 7.5 M.sub.w/M.sub.n 7.6 ρ [g/cm.sup.3] 2.1 T.sub.m/T.sub.c [° C.] 111/94

[0103] The LDPE was cryogenically ground into powder form with particle size ca. 0.5 mm by using a high rotor mill. GNP M25 powder was mixed with acetone and homogenized by means of sonication for 3 h (90 W).

[0104] The well dispersed and homogenized suspension of GNP with acetone was stirred for 20 min by means of rotor-stator mixer Ultra-turrax T 25 IKA at 15 000 rpm in order to get rid of agglomerates in the suspension. Thereafter, LDPE in the powder form was mixed with the GNP-acetone suspension using an overhead stirrer rotating for 40 min at 500 rpm until full evaporation of acetone has been obtained. This process was followed by drying the obtained master batches of composites with various concentration of GNPs at 60° C. for 24 h.

[0105] For experimental verification, different amount of filler material was used in the batches. Specifically, batches were produced having the filler concentration as follows: 5, 10, 15 and 20 wt-%.

[0106] The batch materials were then extruded. The extrusion process of GNP-LDPE nanocomposites was carried out using a circular die. LDPE-GNP master batches were extruded using a Brabender 19/25 D single-screw extruder (Duisburg, Germany) with a compression screw having diameter D=19 mm and screw length of 25×19, and compression ratio 2:1. The extruder was equipped with Terwin 2000 series (model 2076) melt pressure sensors with maximum pressure of 700 bar for inline monitoring. The used temperatures, from the compaction zone, melting and metering zones to the extruder's die, were as follows 115, 130, 130 and 140° C. The used screw speed of 30 rpm was maintained during the whole process.

[0107] The extrusion process was repeated three times. The first two extrusions were treated as a melt compounding of the LDPE-GNP master batches to obtain proper dispersion of the nanofiller within the polymer matrix. Finally, the master batch was extruded into cylindrical extrudates with a diameter of 7 mm. Specimens for antibacterial analysis were collected after the inline pressure reached steady state. Specimens were made with the filler concentration as follows: 5, 10, 15 and 20 wt-%.

[0108] The extruded cylindrical samples were cut in transverse directions T, in a plane perpendicularly to the flow direction, as illustrated in FIG. 2b and FIG. 6, and longitudinally directions L, in a plane parallel to the flow direction containing the axis of the cylinder, as illustrated in FIG. 3b and FIG. 6. The cutting was made with a low speed saw with diamond blade.

[0109] Afterward, the surfaces of the samples were etched or ablated in order to expose the GNP nanoflakes. For this purpose an etchant comprising sulfuric acid, ortho-phosphoric acid and water with 1 wt. % of potassium permanganate was used for 30 h. After the etching/ablating all the samples were carefully cleaned with the mixture of sulfuric acid and deionized water followed by hydrogen peroxide and rinsed twice with deionized water.

[0110] The thus produced samples with antibacterial surfaces were then tested to evaluate the antibacterial activity. The antimicrobial response of extruded and etched/ablated samples of different concentration of GNP-LDPE composites was evaluated by using colony counting method, scanning electron microscopic observation and live/dead viability staining. The antimicrobial activity of extruded samples was tested against the E. coli and S. epidermidis.

[0111] Overnight growth of respective bacterial cultures was diluted to obtain final inoculum of 2-5×106 CFU/ml and seeded on the surface of LDPE (Control) as well as on the LDPE nanocomposite samples containing various concentration of GNP. Bacterial inoculum loaded samples were incubated for 24 h at 37° C. to grow the respective biofilms on the surfaces. After the 24 h, biofilms were rinsed twice with sterile water and collected in 5 ml of 0.89% of sodium chloride. Biofilms were dispersed and homogenized by sonication (30 sec). The homogenized biofilm suspension (100 μl) was serially diluted and plated on agar plate, incubated at 37° C. to count colonies.

[0112] To examine the interaction between GNP nanoflakes and the bacterial cells, biofilms were observed with a scanning electron microscope. The biofilms grown on LDPE surface as well as surfaces with various concentration of GNP were fixed with 3% of glutaraldehyde for 2 h. The fixed biofilms were dehydrated by using graded series of ethanol concentrations (40, 50, 60, 70, 80 & 90%) for 15 min each and with absolute ethanol for 20 min. All the dehydrated biofilms were dried overnight at room temperature and sputter coated with thin layer of gold (5 nm) before SEM imaging. SEM imaging was performed with Supra 55 VP (Carl Zeiss AG, Jena, Germany). To further confirm the bactericidal activity of LDPE-GNP composites, biofilms were stained with LIVE/DEAD® BacLight Bacteria Viability stains kit L7012 (Invitrogen, Molecular Probes, Inc. Eugene, Oreg., USA). The kit contains the green-fluorescent nucleic acid stain SYTO® and the red-fluorescent nucleic acid stain propidium iodide (PI). The green-fluorescent dye (Syto) crosses all bacterial membranes and binds to the DNA of bacterial cells and containing second dye, red-fluorescent PI that only crosses damaged bacterial membranes (dead bacteria). After the staining with mixture of Syto and PI for 20 min, Fluorescence microscopic imaging of the biofilms was performed using a Zeiss fluorescence microscope (Axio Imager.Z2m Carl Zeiss, Jena, Germany).

[0113] From SEM pictures one can see that, GNP nanoflakes are oriented in the polymer flow direction. The SEM pictures of group A, to the left in FIG. 9, show samples cut in a transversal direction at four different GNP concentrations, and the SEM pictures of group B, to the right in FIG. 9, show samples cut in a longitudinal direction, at the four different GNP concentrations. All pictures clearly show the vertical orientation of the GNP nanoflakes.

[0114] The antimicrobial activity of these samples was tested against pathogenic bacteria commonly causing infections: E. coli and S. epidermidis. To assess bactericidal activity of vertically oriented GNP nanoflakes against both Gram-positive and Gram-negative bacteria, E. coli was used as a model organism for Gram-negative bacteria and S. epidermidis was used as a model of Gram-positive bacteria.

[0115] There are a variety of existing strategies for the evaluation of antimicrobial activity. Here we have grown the bacterial cultures on the composite surfaces cut in T (transverse) and L (longitudinal) orientation with respect to direction of extrusion prior to etching/ablating. The reason for the samples being cut in these two ways, has to do with orientation with respect to direction of extrusion and edge-effects. Colony forming units (CFUs) of adherent bacteria were first determined by plating method.

[0116] Significant loss in viability of both E. coli and S. epidermidis was observed on all composite surfaces with GNP regardless of cutting direction, as shown in the diagrams of FIG. 10. The loss in viability was dependent on the concentration of GNP in the GNP-LDPE composites. Even the 5 wt-% of GNP-LDPE composites showed significant bactericidal impact. With T cut samples, the loss of viability was 40±6.5% and 40.8±10.5% against E. coli and S. epidermidis, respectively. In the L cut samples, the loss of viability was 33.55±6.5% and 28.18±7.3% against E. coli and S. epidermidis respectively. The loss in viability of E. coli jumped to 80.8±5.8% and 84.45±11.43% with T and L sliced 10 wt-% of GNP-LDPE composite, and for the same samples, loss in viability of S. epidermidis raised to 58.5±7.4% and 61.12±4.9%, respectively, with T and L sliced sample. This considerable difference in the bactericidal activity of 10% GNP-LDPE composites against E. coli and S. epidermidis might be due the shape of the bacterial cells rather than being gram positive and gram negative bacteria. In the 10 wt-% of GNP-LDPE composites, though the density of oriented GNP nanoflakes appears well distributed and homogenous, there are gaps in between nanoflakes with enough space for circular S. epidermidis cells to attach without interference. Whereas E. coli cells, being rod shaped (with 2 μm length), are more likely to get in contact with the GNP nanoflakes. The loss in viability of S. epidermidis with 15 wt-% of GNP-LDPE was raised to 78.5±8.0% and 99.99±0% with T and L sliced composite sample, whereas bactericidal effect against E. coli remain similar (81.47±3.20%) as for 10 wt-% of GNP-LDPE with T sliced sample. In the case of L sliced sample, loss in viability of E. coli raised to 99.99±0%.

[0117] Moreover, with the 20 wt-% of GNP-LDPE, the loss in viability went up to 90.0±1.7% and 94.5±0.7% of E. coli and S. epidermidis, respectively, with T sliced samples. On the other hand, 99.99% of loss in viability of both E. coli and S. epidermidis was observed with L sliced composite samples.

[0118] The similar loss in viability % of E. coli (gram negative bacteria) and S. epidermidis (gram positive bacteria) observed with the 20 wt. % of GNP-LDPE indicates that antimicrobial efficacy of the material is solely dependent on filler content and geometry of graphite nanoplatelets flakes on the surface, regardless to the different group or type of bacteria. The anti-biofouling activity of the composite material seen with CFUs counting method might be due to bactericidal activity or inhibitory effect on bacterial adhesion, or due to both bactericidal and anti-adhesion activity.

[0119] Moreover, L sliced samples have shown more pronounced antibacterial effect than T sliced. This behavior is probably associated with the fact that the L sliced samples had slightly greater area than T sliced samples, which resulted in higher number of bacteria in contact with GNP flakes for L sliced samples.

[0120] The wetting by the media was also more efficient for the L sliced samples than for the T sliced samples, which also can influence the loss in viability %.

[0121] From these measurements, the following may be concluded: [0122] Even with a low GNP concentration of 5 wt-%, a good antibacterial effect was obtained. [0123] At higher GNP concentrations, at 10 wt-% or above, a very good antibacterial effect was obtained. [0124] At GNP concentrations of 15 wt-%, extremely good antibacterial effects were obtained. [0125] The antibacterial effect was even slightly further improved when 20 wt-% GNP was used.

[0126] Furthermore, to confirm the inhibitory effect on bacterial adhesion and bactericidal effect of GNP-LDPE composites, biofilms formed on control as well as composite samples were stained with live/dead bacterial viability kit and examined under the fluorescence microscope. Using fluorescence probe to detect the viability of bacteria is another widely used method to evaluate antimicrobial activity. SYTO at the concentration of 6 μM is able to cross cytoplasmic membrane and interact with the nucleic acid to enhance the fluorescence giving green fluorescence intensity. Propidium iodide (PI) exhibit a fluorescence enhancement of up to 20- to 30-fold upon intercalation into double-stranded regions of DNA. PI is unable to cross cytoplasmic membrane of bacteria, and consequently generates no fluorescence enhancement in live bacterial cells. In the case of compromised bacterial cell membrane, the used PI interact with nucleic acid and enhance red fluorescence intensity. Hereby, the bacterial cells damaged by vertically oriented GNP flakes are stained red with the PI. The live bacteria in biofilms on control as wells as GNP-LDPE composites are stained green with the SYTO.

[0127] The results of this are shown in FIG. 11, where the pictures of the first four columns are related to T cut samples, and the last four columns to L cut samples. The first two columns of each group, i.e. columns 1-2 and 5-6, are related to E. coli, and the last two columns of each group, i.e. columns 3-4 and 7-8, are related to S. epidermidis. Columns 1, 3, 5 and 7 are showing the live bacteria, originally colored in green but here reproduced in greyscale, whereas columns 2, 4, 6 and 8 are showing the dead bacteria, originally colored in red but here reproduced in greyscale. The first row is directed to the control sample, without GNP, and the last four rows are directed to samples containing 5, 10, 15 and 20 wt-% of GNP.

[0128] As demonstrated in FIG. 11, overall density of biofilms of both E. coli and S. epidermidis were observed to be drastically decreased for the samples containing GNP in the composite compared to the control samples. The decrease in density of bacterial cells in higher concentration of GNP-LDPE composites directly correlates with the concentration of oriented GNP nanoflakes in the composite samples. Interestingly, very few bacterial cells were observed on the vertically sliced 15 and 20 wt. % of GNP-LDPE composite samples suggesting that higher concentration of GNP also plays a role in prevention of bacterial adhesion and as an effect prevention of colonization.

[0129] Further, the interaction between bacterial cells to vertically oriented GNP nanoflakes on GNP-LDPE composite was examined by using a SEM. Since very few bacterial cells were observed in 15 and 20 wt-% L cut samples, only T cut samples were used for SEM analysis.

[0130] The SEM pictures are shown in FIG. 12. Here, the first and second columns are showing E. coli, where the first column has somewhat lower magnification and the second column somewhat higher magnification, and the third and fourth columns are showing S. edipermidis, where the third column has somewhat lower magnification and the fourth column has somewhat higher magnification. Row 1 shows pictures of the control sample, whereas rows 2-5 show pictures of the samples prepared with 5, 10, 15 and 20 wt-% of GNP.

[0131] FIG. 12 demonstrates the contact of bacterial cells to sharp edges of GNP and the damage of bacterial cells by piercing through the cell wall. In the case of lower concentration of GNP in GNP-LDPE composites, a small number of bacterial cells are observed to get in contact with the GNP nanoflakes. In the case of composite samples with higher concentration of GNP, the high amount of vertically oriented GNP nanoflakes enhances the possibility of bacterial cells getting in contact with nanoflakes and leading to the morphological disruption, thereby enhancing the loss in viability. The images from SEM examination follows the results obtained from the colony counting method indicating a higher percentage loss in viable cells in dependence on the concentration of GNP used and the amount of vertically oriented GNP nanoflakes on the GNP-LDPE composite surface.

[0132] The obtained results are consistent with the findings in US 2018/320002, suggesting contact killing or insertion mode of action by graphene. Furthermore, attachment of bacterial cells on the GNP/LDPE composite is observed to be significantly lower with the high concentration (15 wt. % and 20 wt. %) of GNP on composites materials. Prevention in the bacterial adhesion on the samples with higher concentration GNP suggests that the vertically oriented GNP nanoflakes inhibit the biofilm formation by at least two different mechanisms, including the prevention of colonization as well as physical damage of adhered bacterial cells.

Conclusion and Summary

[0133] The results of the experiments clearly demonstrate that controlled orientation of GNP on the polymeric composite materials follows similar mechanism as other graphene derivatives to damage the microbial cells. The results from the antimicrobial assessment show that GNP nanoflakes on the extruded GNP-LDPE composite interact with the bacterial cell membrane physically thereby damaging the cells. This is illustrated schematically in FIG. 13.

[0134] The bactericidal activity is further dependent on the density of oriented GNP flakes on the composite surface, and it was found that particularly advantageous results were obtained when the amount of filler material was 10 wt-% or more. Furthermore, samples with higher concentration of GNP flakes was shown to have strong efficacy to inhibit bacterial colonization. The results show that GNP-LDPE composites can be used to develop e.g. polymer based biomedical devices with bactericidal activity that could prevent possible device associated infections. The production method is fast and cost-efficient, and can easily be scaled for industrial production.

[0135] The invention has now been discussed in relation to different embodiments. However, it should be appreciated by those versed in the art that several further alternatives are possible. For example, the antibacterial coating/surface may be used in many other medical devices, as well as in other applications. For example, the coating/surface may be used in other types of catheters, such as vascular catheters or the like, in other type or irrigation systems, in tubes containing bacteria sensitive contents, in process industry, in packages, etc.

[0136] As discussed in the foregoing, many other materials could be used. For example, it is possible to use other filler materials, with other nanoscale flakes or platelets, than GNP. It is also possible to use other polymer matrix materials than LDPE. It is also possible to use blends of different polymer material as the polymer matrix material. The mixture used for processing may also contain other constituents, e.g. additives such as compatibilizers, stabilizers, antioxidants, plasticizers, etc. The material may also have other antibacterial additives, such as silver or the like. Other ways of removing polymer matrix material at the surface may also be used, in addition to or instead of etching, such as various types of ablation techniques.

[0137] It will be appreciated by those versed in the art that several such alternatives similar to those described above could be used without departing from the spirit of the invention, and all such modifications should be regarded as a part of the present invention, as defined in the appended claims.