Background

Biofilms represent thin organic films which are formed by microorganisms such as bacteria, algae and fungi on a surface or interface. Depending on the function of the respective surface/interface, the adhesion of biofilms may involve very unfavorable economical consequences. For example, microorganisms grow on the hull of marine ships and increase the drag thereof considerably by producing such biofilms. Further examples are the colonization of ventilation systems or medical products. This kind of contamination leads to an increased risk for infections and represents a major cost factor in the health service and gross national product.

Consequently, a wide variety of methods for preventing or reducing the production of biofilms have been developed in the art and include, e.g., mechanical and chemical cleaning processes, application of antimicrobial coatings etc. Most of these methods, however, suffer from one or more disadvantages. For example, cleaning processes usually involve considerable additional costs, antimicrobial coatings are often toxic for both the target microorganisms and other organisms as well and may be inappropriate for specific applications .

Thus, an object of the present invention is to provide alternative methods for preventing or reducing the production of biofilms formed by microorganisms which are cost- efficient, easy to perform and avoid the drawbacks of the prior art.

This object is achieved by providing the method for preparing a nanostructured antimicrobial surface according to claim 1 and the use of said nanostructured antimicrobial surface according to claim 17. Further aspects and/or preferred embodiments of the present invention are the subject of dependent claims.

Description of the invention

The present invention provides a method for preparing an antimicrobial surface which comprises providing a substrate surface with a 3-dimensional nanostructure comprising elevations with a predetermined height in the range of nm, preferably in the range of 10-600 nm, such as 50-600 nm, and a predetermined mean distance in the range of nm, preferably in the range of 10-300 nm, such as 100-300 nm, which is adjusted to be smaller than the size of target microorganisms so that the target microorganisms are not able to penetrate into the space between the elevations.

By preventing the penetration of microorganisms into this interspace and by providing only a considerably reduced or minimal number of contact points (as compared with a non- structured surface) the antimicrobial surface prepared by the method of the invention is adapted to prevent and/or inhibit the formation of a biofilm generated by the target microorganisms .

The target microorganisms may be one or more members from the group comprising bacteria, algae, fungi, protozoae and viruses . The method of the invention for providing a nanostructured antimicrobial surface and the use thereof for preventing and/or inhibiting the formation of a biofilm generated by the target microorganisms represents a novel and innovative concept .

Hochbaum and Aizenberg have merely disclosed that microorganisms such as bacteria pattern spontanously on periodic nanostructure arrays (Nano Letters 2010 10(9), 3717- 3721) . It is also known in the art that tissue cells such as fibroblasts or astroglial cells are affected by the structure of the substrate with respect to their proliferation and tissue-forming properties (Turner et al., Journal of Biomedical Materials Research 2000 51(3), 430-441; Choi et al., Journal of Biomedical Materials Research Part A 2009 89A(3), 804-817). Such tissue cells, however, are clearly different from microorganisms.

The mean distance and maximal distance of the elevations present on said nanostructured surface can be adjusted in such a manner that the target microorganisms cannot penetrate. Since most microorganisms have dimensions in the range from 50 nm to a few micrometers, a maximal distance of about 40-49 nm is suitable to prevent any penetration of such microorganisms. However, for larger target microorganisms, the upper limit of the maximal distance can be varied as appropriate. For example, the majority of microoganisms have diameters above 150 nm, so that a maximal distance of 100-150 nm is suitable for these target microorganisms. Moreover, e.g. viruses may have dimensions of below 10 nm and for preventing the adhesion of these small microorganisms, the elevations can also be provided in a correspondingly denser arrangement . Usually, the elevations have a height in the range of from 10-600 nm, typically 100-300 nm, preferably 200-300 nm, such as about 250 nm, and/or a mean distance in the range of from 10- 300 nm, typically 100-200 nm, preferably 100-150 nm, such as about 150 nm. In the case of highly ordered arrays of elevations, such as those typically produced by a method involving BCLM, the difference between the mean distance of the elevations and the maximal distance of the elevations will often be rather small so that both values are nearly identical or at least of the same order of magnitude.

Thus, the nanostructured surface used in the present invention may be additionally or alternatively defined by the maximal distance of the elevations and said elevations will have a maximal distance in the nm range, such as the range of from 10-300 nm, typically 100-200 nm, preferably 100-150 nm, such as about 150 nm. Suitable upper limits of the maximal distance for specific target microorganisms may be, e.g., < 10 nm, 40-49 nm or < 150 nm.

Preferably, the elevations have an elongated form, wherein the width : height ratio of the elevations is in the range from 1:2 to 1:10, more preferred 1:3 to 1:10. More specifically, the elevations may have the shape of pillars or cones. In the latter case, the width : height ratio relates to the mean width of the cones. Preferably, the elevations have rather sharp tips with a diameter in the range of from 2 nm to 50 nm.

Principally, the material of the substrate surface is not limited and may be any material, in particular any material sitable for nanostructuring by micellar nanolithography. More specifically, the surface is selected from the group comprising metals, metal oxides, silica, glass, organic or inorganic polymers, ceramics. The surface may be planar or curved, such as e.g. present in medical devices, hulls of ships or other constructions, ventilation systems etc. In a specific embodiment of the invention, the antimicrobial surface consists of or comprises the surface of a colloid particle having a diameter in the micrometer range, such as 1-999 μπι, more specifically 5-500 i . Such colloids are, for example, very useful in painting applications and a suspension of such nanostructured colloid particles could be applied to an extended macroscopic surface/interface.

The elevations may be provided on the surface by any suitable method known in the art. The method may for example comprise conventional embossing processes using a stamp or master or a casting or a polymerization process.

In a preferred embodiment of the invention, the method comprises decorating the substrate surface with an ordered array of nanoparticles or nanoclusters by means of micellar nanolithography (BCLM) . In this method, organic templates, e.g., block copolymers and graft copolymers that associate in suitable solvents to micellar core shell systems are used. These core shell structures serve to localize inorganic precursors from which inorganic particles with a controlled size can be deposited that are spatially separated from each other by the polymeric casing. The core shell systems or micelles can be applied as highly ordered monofilms on different substrates by simple deposition procedures such as spin casting or dip coating. The organic matrix is subsequently removed without residue by a gas-plasma process or by pyrolysis as a result of which inorganic nanoparticles are fixed on the substrate in the arrangement in which they were positioned by the organic template. The size of the inorganic nanoparticles is determined by the weighed portion of a given inorganic precursor compound and the lateral distance between the particles through the structure, especially by the molecular weight of the organic matrix. As a result, the substrates have inorganic nanoclusters or nanoparticles , such as gold particles, in ordered periodic patterns corresponding to the respective core shell system used deposited on their surface.

Some non-limiting examples for suitable block copolymers in this method are polystyrene-b-polyethylenoxide, polystyrene- b-poly (2-vinylpyridine) , polystyrene-b-poly (4-vinylpyridine) or mixtures thereof. Preferably, polystyrene-b-poly (2-vinylpyridine) is used. This basic micellar block copolymer nanolithography method is described in detail in, e.g., the following patents and patent applications: DE 199 52 018, DE 197 47 813,

DE 297 47 815, and EP patent No. 1027157. Subsequently the surface decorated with inorganic nanoclusters or nanoparticles is subjected to one or more etching steps, wherein the nanoparticles or nanoclusters serve as an etching mask so that the area covered by said nanoparticles or nanoclusters is protected and elevations, typically in the shape of nanopillars or nanocones, remain at these positions after completing the etching process. The height and shape of the elevations as well as their density or mean distance can be finely adjusted in the nanometer range by selecting appropriate etching conditions and/or further processing steps. Such methods for producing a substrate surface nanostructured with nanopillars or nanocones are for example disclosed in WO 2008/116616 Al, DE 2009 060 223.2 and DE 2010 023 490.7. Fig. 1 shows an exemplary nanostructured glass surface prepared by BCLM and subsequent etching which is suitable for the method of the invention. A microorganism contacting such a surface will have relatively few contact sites and cannot penetrate into the space between the pillars. Thus no stable adhesion/binding of the microorganism, which is essential for the formation of biofilms, can occur.

Optionally, the nanostructured surface which is usually hydrophilic can be made hydrophobic, for example by means of silanizing, preferably by vapour phase deposition. The silane may be any suitable silane known in the art for this purpose. Preferably the silane is selected from the group comprising fluorinated and perfluorinated silanes such as perfluoro- decyltrichlorosilane and structurally related compounds.

This step facilitates the flushing and cleaning of the surfaces and also provides an additional anti-adhesive effect .

In a specific embodiment of the invention, the tips of the elevations are provided with antimicrobial components.

In a more specific embodiment, the antimicrobial components are selected from the group comprising nanoparticles, charged molecules or peptides.

Some non-limiting examples for suitable nanoparticles are particles of Ag, Au, Pd, Pt, Zn0 ₂ , Ti0 ₂ or magnetic particles .

In a preferred embodiment of the invention, the nanoparticles, after subjected to a chemical or physical stimulus, are capable to produce heat or to initiate oxidative processes leading to the disruption of microorganisms, in particular bacteria, in contact with said nanoparticles . More specifically, the stimululus may comprise electromagnetic radiation, such as visible light, UV light, IR, by a conventional light source or laser irradiation, or a magnetic field. Thus, the antimicrobial effect of the nanostructured surface can be enhanced or induced by selecting specific nanoparticles sensible for a desired stimulus and applying specific stimuli. The charged molecules or peptides may comprise any sequences or entities which are suitable to prevent or reduce the adhesion of microorganisms, for examples by interfering with signaling molecules or receptors of the target microrganisms, in particular arginine-rich sequences, lysine-rich sequences or guanidines or biguanidines, such as polyhexamethylen- biguanidine. In some cases, it may be desirable that said peptides or entities are also toxic for eukaryotic cells and suitable peptides or entities are known in the art. The surface to be protected may be any kind of surface which is exposed to undesired microorganisms. In particular, the surface may be part of a medical or dental device, a ventilation system, a mobile or immobile marine construction such as a ship, warp, dock etc., or of a colloid particle having a diameter in the micrometer range.

A further aspect of the present invention relates to the use of a nanostructured surface for preventing and/or reducing the production of biofilms generated by target micro- organisms on surfaces, wherein the nanostructured surface comprises elevations with a predetermined height in the range of nm, preferably 10-600 nm, and a predetermined mean distance in the range of nm, preferably 10-300 nm, which is adjusted to be smaller than the size of the target microorganisms so that the target microorganisms are not able to penetrate into the space between the elevations.

In a specific embodiment of this use, the elevations are nanopillars or nanocones.

The nanostructured surface may be, e.g., advantageously used for preventing and/or reducing the production of biofilms in the fields of medicine, biology, chemistry, construction industry etc.

The invention is further illustrating by the following non- limiting Examples and Figures.

FIGURES

Fig. 1. shows an exemplary nanostructured glass surface used in the method of the present invention

Fig. 2. shows the density of adhering Staphylococcus bacteria on an unstructured flat surface (a) versus a nanostructured surface (b)

Fig. 3. quantitative evaluation of the images shown in Fig. 2; 3A: number of bacteria on each surface; 3B: covered area EXAMPLE 1

Preparation of nanostructured samples of quartz glass

The nanostructuring of the substrate was achieved by following a general working protocol which had been developed in the research group of the inventors and is described in e.g. WO 2008/116616.

Initially, a BCML-solution has been prepared essentially according to the protocol mentioned above (PS-b-P2VP; Mn (PS) =110000, Mn (PVP) =52000, Mw/Mn =1.15, Polymersource, Inc., c=5 mg/ml, L=0,5). The samples have been spin coated with that solution (6000 rpm, 40 i , 1 minute). Afterwards the samples have been subjected to a plasma treatment ( Plasmasystem 100, TePla, 45 minutes, 0,4 mbar, 150W, W10 gas) .

Finally the nanostructures have been etched, using a reactive ion etcher (Oxford Plasma, Plasmalab 80) . The recipe consisting of two steps, which have been repeated 8 times was as follows:

Step 1: Ar : SF6:02 : lOsccm: 0sccm: 8sccm; p:50mTorr; RF- power:120W; t:60s

Step 2: Ar : CHF3 : lOsccm: 0sccm; p:50mTorr; RF-power : 120W; ICP- power:20W; t:20s

In a last step, the samples have been ultrasonicated in ethanol . Both the unstructured flat reference substrates and the nanostructured substrates were provided with a monolayer of a silane, (1H,1H,2H,2H perfluorodecyltrichlorosilane ; from ABCR, Karlsruhe, Germany) via deposition from the gas phase to obtain a hydrophobic surface (deposition conditions: 30 minutes incubation in an evacuated excicator. Afterwards 60 minutes curing in an oven at 80°C under atmospheric pressure) . This step facilitates the flushing and cleaning of the sample plates and also provides an additional anti- adhesive effect.

EXAMPLE 2

Adhesion of Staphylcoccus bacteria to nanostructured substrates versus adhesion to unstructured reference samples

Staphylococcus sciuri subsp. Sciuri (ATCC 29062) obtained from an overnight culture were cultivated in TSBY medium at 37°C and 220 rpm to a density of about 2 x 10 ⁸ cfu/ml. 1.5 ml of this culture were transferred to small quartz glass plates (unstructured/flat or nanostructured) which had been sealed with a silicone ring and were further incubated for 1 h at 37°C without shaking. Subsequently 1.2 ml of the culture was removed and replaced by 1.2 ml medium with DAPI (9 nM) . The plates were further incubated for 15 minutes without shaking at 37 °C. In order to remove medium and non-adhering bacteria, the silicone rings were taken off and the plate surfaces either hosed with 10 x 1 ml lx PBS or dipped 10 times into 1 x PBS.

For microscopy, the plates were fixed on a slide carrier with FixoGum and stored in a humid chamber. Phase contrast and fluorescence images (DAPI (49); Exct . 365 nm, Em. 445/50) of the surfaces were obtained with an Axiovert 200M. Fig. 2 shows the coating of an unstructured flat substrate (a) and of a nanostructured substrate (b) after incubation and flushing. It is clearly visible that the unstructured flat substrate (a) is colonized considerably more densely. Fig. 3 shows the results of a quantitative evaluation using an image processing program (ImageJ) (3A: number of bacteria surface; 3B: covered area (%)).