Patent Application
20070238174
A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
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The present invention relates to novel substrates, to methods of making them and to uses therefor. In particular the invention concerns such materials, methods and uses for the inhibition of cell growth.
The behavior of cells are influenced by their external environment, both chemical and physical. Understanding interactions which take place between a cell and its substrate is important in connection with such fields as medical implants and prostheses, tissue engineering and pharmaceutical development. One substrate characteristic which has been shown to influence cellular behavior is topography and synthetic structured surfaces have been used to investigate this influence. A review of such investigations may be found in Biomaterials (1999), 20, 573-588. In vivo studies are reported in Biomaterials (1996), 17, 2087-2095.
The modification of surface topography for the control of cellular response is an important area of research in medical engineering that targets several potential end uses, particularly relating to the biocompatibility of materials used in medical and dental devices and implants and in the preparation of materials, such as hygienic surfaces, which deter cell growth thereon. In this area, it is required to control the interfacial reactions that mitigate the appropriate response for a specific application. It is known that the interfacial reactions are influenced by the surface properties of the substrate in terms of the surface chemistry, energy and topography. Of the latter, current research is focused on etching techniques to create the desired topography. Experimental Cell Research (1996), 223, 426-435, discusses the production of micro fabricated grooves and steps by means of dry etching a silica substrata with a reactive ion etching unit. U.S. Pat. No. 4,832,759 also discusses the generation of a plurality of surface discontinuities by means of ion beam etching. Many prior art studies have used photolithographic techniques to engineer surface features with controlled morphology for the study of cell behavior thereon. Other techniques include glancing angle deposition, laser ablation, laser deposition, replica molding of x-ray lithography masters, imprint lithography, micro contact printing and etching and ink-jet printing. For example, Canadian Pat No. 2,323,719 discusses the production of structural elevations by the LIGA lithographic process which incorporates x-ray lithography, electrodeposition and molding. Canadian Pat No. 2,302,118 discloses microstructured surfaces produced mechanically or lithographically. DE-A-19818956 discloses materials with a micro-roughened, bacteria-repellant surface. WO-A-97/12966 discloses methods for producing thin colloidal silica films on substrates for growing cells in culture by spin coating or spraying the substrate with colloidal silica, or dipping the substrate in a colloidal silica solution.
Cell-substrate interactions in the natural environment are influenced by the surface topography of the substrate, the topographical features of which are represented at the nanoscale level. Some of the above-mentioned techniques and prior art disclosures for engineering synthetic surface features are capable of generating topographical features at the nanoscale level but none has so far offered a quick and convenient means to inhibit cell growth at this level. Furthermore, the suitability for commercial application of many of the known substrates is limited. One particular problem with some prior art substrates is the tendency of the micro-structured surface layer to crack or peel.
It is an object of the present invention to provide a substrate which can be used to inhibit cell growth at the nanoscale level. It is a further object of the invention to provide such a substrate which is sufficiently robust to be useful in commercial applications. Another object of the invention is to provide a substrate which can be manufactured reproducibly, conveniently and without excessive expense.
According to the present invention there is provided a cell growth inhibiting substrate comprising:
a base portion;
a surface layer covering at least part of the base portion, the surface layer providing the substrate with topographical features having at least one nanoscale dimension of from about 1 to about 200 nm; and
a binding layer between the base portion and the surface layer for binding the surface layer in place.
The substrate has been shown to inhibit the growth of a number of different cell types, ranging from mammalian cells, fungal cells (including yeast) and bacterial cells. The substrate of the invention therefore provides a novel material which may find application in a wide variety of circumstances. For example, substrates according to the invention may be used to provide medical implants whose surfaces discourage cell growth. Hygienic surfaces, for use in hospitals, restaurants, kitchens, bathrooms etc. may be formed from substrates according to the invention. Furthermore, existing surfaces may be modified to form the substrates of the present invention. Prior art substrates having nanoscale topography have tended to be unsuitable for many commercial applications as they are not sufficiently robust to withstand exposure to the conditions of such applications. Many prior art substrates have nanoscale topography which cracks, flakes or peels over time, rendering the substrate unsuitable for most applications other than short term academic study.
The term, “inhibit”, “inhibition”, or “inhibiting” in relation to cell growth is defined herein as limiting or preventing cellular proliferation.
In the context of this document, “nanoscale” is used to refer to topographical features having at least one dimension which is measurable at the nanometre level, for example a feature which measures from about 1 to about 200 nm, preferably from about 1 to about 150 nm, even more preferably from about 1 to about 100 nm, and most preferably less than about 50 nm in at least one dimension.
The topographical features may form a random array on the surface layer of the substrate. Such an array may comprise, for example, an agglomeration of peaks and troughs, preferably having substantially the same or similar dimensions and physical characteristics.
Alternatively, the topographical features may form an ordered array on the surface layer of the substrate. A combination of random and ordered arrays may be used also. Whatever form of array adopted by the topographical features, the substrate of the invention preferably comprises nanoscale topographical features separated from other nearest neighbor similar nanoscale topographical features by distances of up to about 1000 nm. For example, when the array comprises individual peaks and troughs, each peak in the array may be separated from its nearest neighbor peaks by distances of up to about 500 nm, preferably no more than about 200 nm. Where the array comprises a series of longitudinal ridges, each ridge in the array may be separated from its nearest neighbor by distances of up to about 1000 nm, preferably no more than about 500 nm.
Preferably the base portion and the surface layer are of different materials and preferably the material of the binding layer is different from one or both of the materials of the base portion and the surface layer. The binding layer may comprise a single layer comprising one or more materials, provided that the binding layer comprises at least one material capable of binding to the base portion and at least one material capable of binding to the surface layer. The same material of the binding layer may bind to both the base portion and to the surface layer, in which case the binding layer may comprise a single material. However, the binding layer may comprise a plurality of materials and may be a composite layer. Thus, for example the binding layer may comprise two layers containing, respectively, a first material and a second material. The first material may be capable of binding to the base portion and to the second material. The second material may be capable of binding to the first material and to the surface layer. The substrate of the invention may comprise additional layers located between the surface layer and the base portion. Such additional layers may comprise one or more bilayers of surface layer material and binding layer material.
Further provided in accordance with the invention is a use of a substrate according to the invention for the inhibition of cell growth. The present invention has been shown to inhibit the growth of a number of cell types. The examples described later show inhibition of mammalian cells (fibroblast and epithelial cells), bacterial cells (Staphylococcus aureus, Psuedomonas aeruginosa and Streptococcus mutans), and fungal cells (Aspergillus niger, Candida albicans and Aureobasidium pullulans).
The topographical features are preferably provided by means of controlled deposition onto the base portion of a surface layer capable of adhering to the substrate. Such adherence may be chemical or physical.
Thus, in one of its aspects this invention relates to methods for tailoring the surface topography of substrates using the controlled deposition of thin films of nanoscale material onto an underlying base portion so as to inhibit cell growth on the treated surface. For example, the substrate may be used in the manufacture or treatment of a hygienic work surface (such as in a restaurant), the surface of a fluid conduit (such as a beer delivery tube or air conditioning duct), or even a medical implant (such as an intraocular lens).
The present invention further provides a method for manufacturing a substrate useful for the inhibition of cell growth comprising the steps of:
a) providing a base portion, a material suitable for forming a surface layer on the base portion, and a binding material suitable for forming a binding layer between the base portion and the surface layer;
b) contacting the base portion with the binding material under conditions effective for at least partial binding of the binding material to the base portion; and
c) contacting the at least partially bound binding material with the surface layer material under conditions effective for at least partially binding the surface layer to the binding material to form a surface layer at least partially covering the base portion, the surface layer comprising topographical features having at least one nanoscale dimension of from about 1 to about 200 nm.
If necessary, the method may further comprise the step of completing the binding of the binding material to the base portion and/or the surface layer.
Also provided in accordance with the invention is a method for manufacturing a substrate useful for inhibition of cell growth comprising the steps of:
a) providing a base portion, a material suitable for forming a surface layer on the base portion, and a binding material suitable for forming a binding layer between the base portion and the surface layer;
b) contacting the surface layer material with the binding material under conditions effective for at least partial binding of the binding material to the surface layer material; and
c) contacting the at least partially bound binding material with the base portion under conditions effective for at least partially binding the base portion to the binding material to form a surface layer at least partially covering the base portion, the surface layer comprising topographical features having at least one nanoscale dimension of from about 1 to about 200 nm.
If necessary, the method may further comprise the step of completing the binding of the binding material to the base portion and/or the surface layer.
Thus, in one of its aspects the invention provides a substrate, modified with a surface of deposited nanoscale material in order to control and inhibit the cellular response that occurs as a result of cell contact or interaction. Further provided is a process of tailoring surface topography by the deposition of nanoscale material onto an underlying substrate material so as to inhibit the cellular response that occurs as a result of cell contact or interaction with that surface.
In one preferred embodiment of the invention there is provided a cell growth inhibiting substrate comprising, a base portion being provided on a surface thereof, by means of controlled deposition onto the base portion, of a substance capable of adhering to the base portion, with topographical features having at least one nanoscale dimension and a cell or tissue growth inhibition region thereon and/or therebetween, the topographical features being deposited in a densely packed array with a separation between nearest neighbor similar topographical features of not more than 1000 nm.
In another preferred embodiment of the invention there is provided a method of manufacturing a substrate for the inhibition of cell growth comprising, providing a base portion, depositing onto the base portion a substance capable of adhering thereto in order to provide the substrate with topographical features having at least one nanoscale dimension the deposit being densely packed with separation between the topographical features of not more than 1000 nm, and providing the substrate with a cell or tissue growth inhibition region on and/or between the topographical features.
The base portion may be selected from any suitable material, depending for example on whether it is intended to inhibit or control cell or tissue growth on the base portion material itself or only on the covering surface layer. The end use of the substrate may also help determine the choice of base portion material, a relatively rigid material being used in the manufacture hygienic work surfaces, for example. The base portion may comprise a single material or may comprise two or more layers of different materials. The base portion is preferably formed from a material selected from polymers, glasses, ceramics, carbon, metals, composites and paper (including tissue paper). The base portion may also comprise an existing surface that is modified in accordance with the present invention. This may for example be an existing work surface or swimming pool surface, to which a surface layer is applied.
The surface layer is preferably formed from a material selected from polymers, glasses, ceramics, carbon, metals and composites. Suitable surface layer materials include silica, gold and silver. The surface layer may comprise colloidal particles of these or other materials and such colloidal particles may be nano-particulate, for example having mean diameters of from about 5 to about 80 nm.
The binding layer may comprise one or more substances capable of adhering to the base portion and the surface layer material. Suitable binding layer materials include polymers, surface active agents, reactive chemical ligands and polycationic materials. Preferably the adhering substance is insoluble or sparingly soluble. Inorganic, organic, metallic and polymeric materials, or mixtures of one or more thereof, may be used.
The substrates, methods and uses of the invention have advantages over conventional engineered surfaces used in medical engineering and methods for making them. The growth of different cells can be inhibited by selecting different topographies, as cells respond differently to different physical environments. The invention therefore provides a valuable tool for use in medical engineering. The substrates of the invention may also be adapted to provide robust surfaces for use, for example, in hospitals, restaurants and kitchens where it is desirable to discourage eukaryotic and prokaryotic cell growth on surfaces such as bench tops, walls, floors and fluid delivery tubes.
The substrate may be applied to, or incorporated into a wide variety of products and surfaces. For example, the substrate may be used in the following list (which is by no means exhaustive) of products:
It will be apparent to the skilled person, that the substrate of the invention can be manufactured using materials which are non-toxic to human health. For example, the use of amorphous colloidal silica (SiO2— silicon dioxide) is known to be non-toxic to humans. Other non-toxic colloidal materials may also be employed, such as gold or silver.
The substrate may be applied to an existing product or surface by a number of methods, such as spraying or washing. These methods are particularly suited to products or surfaces having complex geometries (such as dentures plates) or surfaces which are inaccessible (such as the interior of beer delivery tubes).
If necessary, the substrate may be manufactured from materials which allow the substrate to be flexible so that it can be applied to, or integrated with products which are flexible or allow for a certain degree of flexibility.
The invention will now be more particularly described with reference to the following Figures and Examples in which:
Substrates in accordance with the present invention are prepared generally with a base portion, a binding layer, and a surface layer. The base portion provides support as well as a suitable surface area for the application of the binding layer and surface layer. Typically, the base portion of the novel substrate is selected with characteristics appropriate for the environmental conditions of which the substrate will be subjected and is generally formed from polymers, glasses, ceramics, carbons, metals, composites, papers, and cellulosic materials.
A preferred base portion for the substrate is polystyrene which is a polymer comprised of the monomer styrene (vinyl benzene) and advantageously can be melted and molded or extruded and then resolidified to form a desired shape for the base portion. Polystyrene is often utilized in common laboratory equipment comprising such articles as test tubes and Petri dishes, typically formed from injection molding. Advantageously, the substrate of the present invention may utilize polystyrene thus imparting novel properties on a material proven capable for a variety of research and diagnostic tasks. Yet furthermore, while polystyrene is a preferred base portion of the substrate of the present invention, the base portion may also comprise other polymers including other thermoplastics such as polyethylene, polypropylene, and nylon; thermosets such as vulcanized rubbers, bakelite, and resins; elastomers such as polybutadiene, polyisoprene, and other saturated and unsaturated rubbers; and other rigid or semi-rigid polymeric materials.
In further preferred embodiments of the substrate, the base portion may comprise ceramics and glass, ideally suited for sanitary ware, medical implants, work surfaces, as well as other uses wherein ceramics and glass are generally known to be utilized. More specifically, ceramics for the base portion may include white ware ceramics such as porcelain useful for comprising toilets, baths, basins, showers and respective fittings.
Addition base portions for the substrate including but not limited to metals, carbons, papers and cellulosic materials may be utilized, thus providing a variety of materials and compositions with desirable cellular response characteristics. With these materials as well as polymers and ceramics and glass, multiple types of materials may be combined in layers to comprise the base portion of the substrate.
The binding layer of the substrate of the present invention may comprise one or more materials capable of adhering to the base portion as well as providing a bonding surface for the surface layer. The binding layer may include but is not limited to polymers, surface active agents, reactive chemicals, ligands and polycationic materials. Preferably the binding layer is insoluble, substantially insoluble, or sparingly soluble. One selection of cationic materials useful for creating the binding layer of the substrate includes Zetag™ of Millienium Technologies Ltd. Further binding materials may include inorganic, organic, metallic and polymeric material and combinations thereof for binding the surface layer to the base portion of the novel substrate.
Yet furthermore, the binding layer may include one or more layers, and further may include a plurality of types of materials. The first layer of the binding layer may be capable of binding to the base portion and to the second layer of the binding layer whereas the second layer may be capable of binding to the first binding layer and to the surface layer. Additionally, additional binding layers may be included between the first and the second binding layers.
The surface layer comprises a nanoscale material which may impart cellular control characteristics to the novel substrate. Generally, the surface layer is selected from materials including polymers, glasses, ceramics, carbons, metals and composites and preferably includes silica, gold, silver or combinations thereof. The surface layer may comprise particles of these or other materials with an average diameter of from about 5 nanometers to about 80 nanometers thus forming a colloidal surface layer. Preferably amorphous colloidal silica may be utilized as it is known to be substantially non-toxic to humans.
In order to create the novel substrate, a suitable base portion is first selected based on the desired use and environmental condition to which the substrate will be subjected. Preferably, the base portion is washed with methanol and water prior to application of the binding layer.
The binding layer may be subsequently be applied to the base portion, in a variety of manners, including both immersion and spray-treating so as to fully apply the binding layer to the desired surface for the cellular control properties. Advantageously, spray-treating of the binding layer may be used in conjunction with base portions having complex geometries as well interior surfaces for which immersion would not be as efficient. Multiple layers applied through similar or different techniques may be applied to the base portion, depended on the chosen binding layer materials as well as eventual application of the substrate. One category of binding layers includes cationic polymers which may function as to ionically bind the particles of the surface layer to the binding layer of the substrate.
The surface layer may also be applied to the combination base portion and binding layer through either immersion, spray-treating, or through other application techniques known in the art for applying colloids. Generally, the colloid comprises of from about a 5% to about a 85% colloidal particle solution, and more preferably of from about a 20% to about a 40% colloidal particle solution. Furthermore, the colloid characteristics are determined by particle type and size as well as the desired application of the finished substrate. Preferably, a silica colloid may be utilized in conjunction with a cationic binding layer, which should result in the formation of a ionically bound monolayer.
A substrate formed from the aforementioned steps may initiate a distinct cellular response affecting the both the morphology and adhesion of cells to the surface of the substrate this limiting cellular proliferation. Cell adhesion may be precluded or limited in both eukaryotic and prokaryotic cell types. In order to further illustrate the principles and operation of the present invention, the following examples are provided. However, these examples should not be taken as limiting in any regard.
Three 10×10×1 mm2 optically clear polystyrene (PS) squares are cut out from a plasma treated polystyrene culture dish. Each PS segment is then washed once with methanol followed by copious rinsing with deionized water (Millipore-Q 18.2 M). Each cleaned PS sample is then half immersed in an aqueous solution of 1 g L−1 polycationic polymer (Zetag)™ for approximately 15 minutes to allow for the development of a monolayer of polycationic polymer on the PS surface. The polycationic derived PS samples are then removed from the polymer solution and washed copiously with deionized water (Millipore-Q 18.2 M) to remove excess polycationic polymer. The coated portions of each PS sample are then immersed in three different aqueous dispersions of silica solution (Ludox TM-50; HS-40 and SM-30, ex. Dupont de Nemours & Co.) containing approximately 40% w/w silica particles of approximately 21, 14 and 7 nm diameter respectively. A further aqueous dispersion of silica solution is also used which contains approximately 10% w/w silica particles of approximately 80 nm in diameter. The surfaces of each silica coated PS sample are then scanned using atomic force microscopy (AFM). An AFM image of the surface of a 21 nm coated sample is shown in
The samples of silica coated PS are then used as substrates in cell culture experiments. A suspension of clone L 929 mouse fibroblast established cell line is prepared from a culture maintained in Eagle's Minimum Essential medium with a 5% foetal calf serum supplement. The suspension is prepared at a cell concentration of approximately 1×105 cell/ml. This is performed by immersing each PS sample in a cell culture medium containing established fibroblast cells for approximately 24 hours in an incubator at 37° C.
After this period the PS samples are removed from the culture medium and examined with an inverted phase-contrast light microscope. An image observed on 14 nm silica coated PS is shown in
In a variant of the previous experiment, the cell suspension is only applied to the untreated substrate and the cells are allowed to spread to the interface during a 48 hour incubation period.
In a variant of the previous experiment, the cell suspension is only applied to the untreated substrate and the cells are allowed to spread to the interface during the 48 hour incubation period. As can be seen in
In order to assess the growth characteristics of different cells on the substrate, bovine lens epithelial cells are applied to the glass substrate partially coated with nano-particulate material.
Primary bovine lens epithelial cells are obtained from the Unit of Opthalmology, The University of Liverpool at second or third passage and maintained in Dulbecco's Minimal essential Medium supplemented with 10% foetal calf serum. A cell suspension is prepared at a cell concentration of approximately 5×104 cells/ml. 1 ml of this cell suspension is directly applied to both treated and untreated surface of a substrate prepared as described in Example 1. The cells are left in contact with the substrate for 30 minutes to allow cells adhesion, then the substrate is flooded with culture medium and maintained at 37° C./5% CO2 for 48 hours. After this time the culture medium and non-adherent cells are removed. The substrate is then treated with 100% methanol in order to fix the cells and the substrate is stained with 0.04% methylene blue for 10 minutes.
In conclusion, epithelial cells are shown to behave in a similar manner to L 929 fibroblast cells on the treated and untreated substrate and it could also be postulated that other cell types will behave in a similar manner.
PMMA and similar materials are often modified with an air glow discharge plasma in order to improve their wettability. Therefore an experiment will be conducted in order to assess the growth of fibroblast cells on a PMMA substrate which will be treated with an air plasma.
A suspension of clone L 929 mouse fibroblast established cell line is prepared from a culture maintained in Eagle's Minimum Essential medium with a 5% foetal calf serum supplement. The suspension is prepared at cell concentration of approximately 1×105 cell/ml. 1 ml of the cell suspension is directly applied to the surface of an air plasma treated polymethylmethacrylate substrate which is prepared according to a standard protocol. The cells are left in contact with the substrate for 30 minutes to allow cells adhesion, then the substrate is flooded with culture medium and maintained at 37° C./5% CO2 for 48 hours. After this time the culture medium and non-adherent cells are removed. The substrate is then treated with 100% methanol to fix the cells and the substrate is stained with 0.04% methylene blue for 10 minutes.
The L929 fibroblast cells are then tested on a PMMA substrate following air plasma treatment and subsequent nano-particulate coating.
The method is the same as outlined above and the cells are directly applied to the surface of an air plasma polymethylmethacrylate substrate as described in Example 1 with a subsequent nano-particulate coating.
The cell growth modifying effects of a layer of silica nano-particles on PMMA, polystyrene and glass will be assessed in Examples 1 to 3. Further studies are directed to alternative nano-particles which could be used, one such material was gold.
A gold particulate preparation of a 30 mM aqueous solution of hydrogen tetrachloroaurate (Aldrich) is mixed with 80 ml of a 50 mM solution of tetraoctyl ammonium bromide (Fluka) in toluene (AnalaR grade) forming a two phase mixture. The organic layer containing the [AuC14]− and [N(C8H17)4]+ ions is then washed three times with de-ionized water. 25 ml of freshly prepared 0.4 M aqueous sodium borohydride (Fisons) is then added in small aliquots with vigorous stirring. The resulting colloidal gold solutions (approx. 5 nm in diameter) are deployed using the same procedure describe for the use of silica particles in Example 1.
Additional experiments are directed to non-mammalian cells. Staphylococcus aureus is chosen to investigate whether the nano-particle substrate could alter growth characteristics of prokaryotic cells.
A strain of S. aureus bacteria is prepared in full growth broth then placed directly in contact with a plain glass base substrate. A 1 ml suspension of the bacteria is presented to the substrate at a concentration of approximately 107 cells/ml The bacterial will remain in contact with the substrate for 60 minutes. The substrate is then washed with distilled water and prepared for scanning electron microscopy by fixing in 2.5% gluteraldehyde and dehydrating in a series of alcohols.
A further experiment is conducted that will utilize the same strain of S. aureus cells and will be provided to a slide by the same procedure as outlined above. The S. aureus will be presented to plain glass substrate coated with Au organosol which will be prepared in accordance to the protocol outlined in Example 4.
An experiment is conducted to assess whether treating ceramic tiles and glass with colloidal silica will inhibit the growth of Aspergillus niger.
A tile board is treated with glacial acetic acid to simulate aging of the grout and tile surface then treated with the various coatings of Zetag™, Zetag™ plus 7, 14 or 21 nm silica or untreated ceramics as shown in
After 7 days, fungal growth is evident with dark staining on the ceramic tiles, which highlighted the growth of fungal spores on untreated and Zetag™ coated tiles. On the silica coated tiles, it is noted that fungal growth is significantly less or absent. Less dark staining is apparent on 14 and 21 nm silica coated tiles and hence less spores adhere to the surface.
In
Parallel studies on glass are carried out to permit microscopic examination of fungal growth. Using glass coverslips coated with a commercial cationic polymer (Zetag™), anionic colloidal silica particles of sizes 7, 14 and 21 nm are deposited by dipping the slides into aqueous silica solutions.
An experiment is conducted to assess whether treating a Petri dish with silica will inhibit the growth of Candida albicans.
C. albicans adhesion and surface associated growth is observed on tissue culture polystyrene (TCPS) Petri dishes (90 mm diameter) which will be partially coated with 7, 14 or 21 nm silica exposing and untreated portion of the same dish. The lid of the Petri dish is removed and placed underneath it providing a sloping surface so that the dish could be partially coated with Zetag™ and a monolayer of silica. Partial coating of the Petri dishes is carried out using a two step procedure as follows:
1. 15 ml of Zetag™ (3.2 g/L) is placed into the dish to partially coat the surface and is left for 10 minutes before being removed, rinsed with water and allowed to air dry; and
2. After drying, the Petri dishes are then partially immersed in 15 ml of silica (7, 14 or 21 nm diameter) before being further rinsed and allowed to air dry (
C. albicans is maintained, grown and subcultured on Potato dextrose agar and stored at 4° C. Single colonies of C. albicans are propagated overnight while shaking at 37° C. in 10 ml of yeast peptone glucose (YPG) media (2% w/v D-glucose, 1% w/v yeast extract, 1% w/v malt extract and 0.1% w/v bacterial peptone). The overnight culture is diluted to an optical density (OD) of 0.1 at 550 nm in sterile YPG containing 5×107 cell/ml. Horse serum is added to the YPG/cell suspension (10% v/v) to encourage hyphal development. 10 ml of the C. albicans culture is transferred aseptically on to partially coated Petri dishes and cultured for 24 h at 37° C. The Petri dishes are rinsed with 10 ml of sterile phosphate buffered silane (PBS) before being fixed with 4% (w/v) glutaraldehyde and stained with 0.4% (w/w) crystal violet.
After 24 h in culture, the untreated portion of the dish will show a mixture of two distinct cell types. A high density of rounded budding yeast cells is apparent and highly branched cells, which represent hyphal development (
Candida induced stomatitis is a considerable problem for immunocompromised denture wearers. Stomatitis is normally treated by antifungal drugs. However, the development of a Candida infection may be prevented by applying a coating similar to the one described above and this would hopefully negate the need for drug therapy.
A further fungal species Aureobasidium pullulans is used to investigate surface growth after 3 days in humid incubation at 25° C. on glass microscope slides coated with 7 nm silica (
Untreated glass control surfaces (
An experiment is conducted in order to investigate surface modification using nanoparticulate silica to reduce biofouling of surfaces.
Three bacterial species are used to test nanoparticulate silica coated surfaces, firstly by rinsing the surfaces to demonstrate their ease of removal and secondly, to monitor the effect of nanoparticulate silica coated substrates upon colonization and subsequent growth using the following test microorganisms: Staphylococcus aureus, Psuedomonas aeruginosa and Streptococcus mutans.
Microbial retention assays are carried out on untreated, Zetag™ treated and silica coated 1 cm2 polyethylene tetrapthalate (PET) substrates following methods of Hirota and co-workers (Hirota, et al., FEMS Microbiology Letters. 248, 37-45, 2005). Overnight cultures of S. aureus and S. mutans will be prepared using 10 ml of bovine heart infusion (BHI) containing 1% glucose, and for P. aeruginosa 10 ml of tryptone soya broth (TSB) containing 1% glucose. Cell cultures are centrifuged and washed three times in 10 ml PBS to obtain cell suspensions for seeding. Cell suspensions of 0.5 ml of S. aureus, P. aeruginosa and S. mutans (1×109 cells/ml) are placed in to 24-well plates containing control, Zetag™ treated and 7, 14 or 21 nm silica coated PET substrates and incubated at 37° C. over a 2 h (
The cell suspension is removed from each PET sample as well as the plate and transferred into a fresh, sterile 24 well plate. Each sample is rinsed three times with 1 ml of sterile PBS, which is collected in a universal container for viable cell counting on BHI and TSB agar plates to calculate the number of viable cells (counting colony forming units—CFUs) removed during rinsing of each surface. Furthermore, untreated, Zetag™ treated and 7, 14 or 21 nm silica coated PET samples are transferred into a universal container are 3 ml of sterile PBS is added and vigorously vortexed and agitated on an eppendorf mixer for 30 sec. The remaining supernatant is used for viable cell counting (colony forming units—CFUs) to estimate cell numbers adhering to each surface.
In
Advantageously, a substrate having a surface profile with nanometer scale dimensions is provided and created with relatively lesser costs than etched or ablated materials. The novel substrate may initiate a distinctive cellular response affecting the morphology, adhesion, and proliferation of cellular material not hereforeto seen.
The disclosures of all cited patents and publications referred to in this application are incorporated herein by reference.
The above description is intended to enable the person skilled in the art to practice the invention. It is not intended to detail all of the possible variations and modifications that will become apparent to the skilled worker upon reading the description. It is intended, however, that all such modifications and variations be included within the scope of the invention that is defined by the following claims. The claims are intended to cover the indicated elements and steps in any arrangement or sequence that is effective to meet the objectives intended for the invention, unless the context specifically indicates the contrary.
This application is a Continuation-In-Part application which claims benefit of co-pending U.S. patent application Ser. No. 10/480,780 filed Jun. 11, 2002, entitled “Substrates”, which was a National Phase filing of International Application No. PCT/GB2002/002652 filed Jun. 11, 2002, entitled “Substrates”, both of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10480780 | Jun 2004 | US |
| Child | 11708660 | Feb 2007 | US |
