Patent Application
20110183867
This invention relates to a method of screening arrays of polymers having pre-determined surface energies. The polymer arrays of the present invention can be used to screen for microorganism adherence. More specifically the arrays can be used to screen for adherence of particular bacteria or fungi to particular polymers in the array. Furthermore, this invention relates to a method combining in-situ polymer synthesis with physico-chemical characterisation of the resulting polymer array and subsequent biological assays of bacterial or fungal adherence. This allows for high throughput screening and characterisation of candidate polymers which are not susceptible to bacterial or fungal adherence or which can be used to support bacterial or fungal adherence where such is required. The arrays can also be used to screen for inhibition or promotion of biofilm formation.
The surface controls many important material performance properties such as biocompatibility and wettability. Surface properties cannot be assumed from bulk properties and thus are usually only determined by direct measurement. Important surface properties include wetting, frictional resistance, wear resistance, absorptivity, adsorption, brightness and luminescence.
Surface energy (γ) is a fundamental property of surfaces and is defined as the energy required to form an additional unit area of surface. It has been shown to correlate with a wide range of surface phenomena such as wetting, adsorption and bioadhesion. Surface energy can also be considered to be a measure of the attractive forces between the molecules of a surface and a liquid. For most surfaces this attractive force is made up of two types of contributing forces: disperse and polar. The dispersive component is that due to London van der Waals forces which operate between all substances (polar and non polar), whereas the polar component is due to more discrete interactions such as hydrogen bonds. Hence, for example, a saturated hydrocarbon would be expected to have zero polar contribution to γ because it is purely disperse. Surface energy may be estimated using a number of experimental methods, including atomic force microscopy, surface force apparatus and inverse gas chromatography. The most common method of γ estimation is by contact angle measurement, which can be achieved using a variety of known methods.
It is known that microorganisms such as bacterial cells are often able to adhere to surfaces and that the ability to develop polymeric surfaces that are largely free of microorganisms can help reduce either the likelihood of infection in medical applications or cross contamination in other situations. Relevant microorganisms include, without limitation, bacteria (both Gram negative and Gram positive organisms) and fungi (eg Candida spp. and Aspergillus spp.). Medical devices where such polymers would be suitable include, without limitation, catheters, shunts, heart valves, corneal implants, and prosthetic joints. There are a number of other medical and non-medical applications for the polymers with non-microbe adherent surfaces. In addition, there are some applications where the selection of polymers which promote micro-organism adherence and biofilm development using the process of the present invention would be advantageous e.g. for the development of stable biofilms in fermenters for the production of useful metabolites and recombinant proteins, for biofilm in systems designed for purification or extraction of useful or harmful substances. The invention is thus also directed towards this goal.
The rate of materials development can be limited by the length of time it takes to produce and test new materials. One approach to accelerate this process is to produce an array of materials and assess them in parallel.
U.S. patent application Ser. No. 10/214,723 discloses the production of polymeric microarrays and the seeding of the biocompatible polymers with cells. This document does not, however, refer to any use of surface analysis techniques to tune the surface chemistry of the polymers.
Anderson et al, Nature Biotechnology (2004), volume 22(7), 863-866 discloses a minaturised cell compatible array of polymers and investigates the effects of the biocompatible polymers on human embryonic stem cell growth and differentiation. This document does not refer to avoidance of adherence or non-human cells.
US2002/0142304 describes a microarray of polymeric biomaterials on a cytophobic surface and the use of the microarray in a screening method. The screening method of the document is intended to screen for the ability of the biomaterials to affect cellular behaviour. The patent refers to the ability to control cellular behaviour eg adherence, proliferation, differentiation, gene expression for a number of unspecified applications. The arrays are intended to investigate the effect of a variety of polymeric biomaterials on a variety of aspects of cellular behaviour. The arrays are also said to be useful for investigating the effect of a variety of natural and synthetic compounds such as drugs, growth factors, proteins, polysaccharides, polynucleotides, lipids, etc on cellular behaviour. The patent describes a wide variety of cell types and a wide variety of polymers but is concerned in particular with mammalian cell properties. There is no disclosure of the concept of preparing an array with a deliberately varied range of surface energy values or the use of such an array to probe bacterial adherence in particular. Furthermore, the document does not recognise the issue of biofilm formation or the problems associated with biofilm formation on surfaces.
Prior art polymeric microarrays are created and seeded with cells without forming a detailed understanding of the surface energy. There is no disclosure in any of the prior art on how producing an array with a wide range of surface energies can assist in identification of polymers with suitably low microbial adherence.
It is an aim of the invention to overcome the various disadvantages of the prior art. More specifically, is an aim of the invention to provide a high-throughput method of creating polymers so that their surface properties, such as surface energy, can be correlated with the desirable effect of low microbial surface adherence. It is a further aim of the invention to provide polymers that have low microbial surface adherence. It is another aim to provide polymers which are resistant to biofilm formation. It is a further aim to produce polymers which promote development of stable biofilms.
The processes and compositions of the present invention satisfy some or all of the above aims.
We have created a microarray of polymers on a substrate which can be used to screen different bacteria or fungi both for the presence of little or no microbial adherence and also for detecting polymers which promote microbial adherence and biofilm development. We have also characterised the surface properties of the individual polymers in the microarray using new data capture procedures based on automated X-ray photoelectron spectroscopy (XPS) secondary ion mass spectrometry (SIMS) and water contact angle measurement.
In our process, picolitre spots of premixed monomer or monomers are introduced on to a slide and irradiated with UV light in order to initiate polymerisation of the monomer combinations in individual spots.
Some of the materials identified by the array are particularly suitable at resisting bacterial adherence. Cells are brought into contact with the array and then probed by a technique such as fluorescence imaging.
In one aspect, the invention relates to a process for screening an array containing two or more different synthetic polymers, the process comprising:
The array can then be seeded with a microbial culture.
In a further step, the microbial adherence onto the polymeric element of the array is monitored by detection of the microorganism to identify polymers with low microbial adherence. Alternatively, the monitoring is performed to detect polymers with a high microbial adherence.
According to another aspect of the present invention, there is provided a process for screening an array containing two or more different synthetic polymers, the process comprising:
Thus, as an alternative step (f) to replace step (f) in the above process involves monitoring microbial adherence onto the polymeric element of the array by detection of the microorganism to identify polymers which promote microbial adherence. These polymers find utility in forming biofilms etc.
In an embodiment, the surface energy of the individual polymer elements is determined by contact angle measurements.
In an embodiment, some or all of the plurality of individual aliquots include more than one type of monomer in the individual aliquots. Thus an individual aliquot may include two or more monomers.
In an embodiment, a proportion of the monomers is liquid at room temperature.
In an embodiment, a proportion of the liquid monomers is provided in a solvent.
In an embodiment, the step of exposing the plurality of aliquots to initiating conditions involves introducing an initiator to some or all of the plurality of individual aliquots using a liquid handling device, and preferably using a robotic liquid handling device.
In an embodiment, the initiator can be an organic radical initiator or a redox initiator.
In an embodiment, the step of exposing the plurality of aliquots to initiating conditions involves exposing the array to electromagnetic radiation and/or thermal radiation, optionally in the presence of an initiator which has been introduced into some or all of the plurality of individual aliquots. The electromagnetic radiation may be UV light.
In an embodiment, the initiator which is present in different individual aliquots is the same initiator. Equally, different initiators could be used for different aliquots.
In an embodiment, the substrate comprises a material selected from the group comprising: glass, ceramic, metal and plastic or a combination of one or more of these.
In an embodiment, the surface of the substrate has been modified to improve retention of the plurality of individual aliquots.
In an embodiment, the surface modification is provided by plasma etching, a polymer coating, chemical treatment or a combination of these.
In an embodiment, the monomers are monomers of polymers independently selected from the group comprising: substituted polyacrylates, substituted polyethers, substituted polycarbonates and substituted polyanhydrides. Some or all of the resulting polymers are biocompatible.
In an embodiment, the polymer includes a degree of unsaturation.
In an embodiment, each individual polymer is independently selected from the group comprising: monofunctional acrylate esters, polyfunctional acrylate esters, monofunctional methacrylate esters and polyfunctional methacrylate esters.
In an embodiment, each individual aliquot has a volume of 500 picolitres or less, and preferably 100 picolitres or less. The individual aliquots may be 50 picolitres or less. The individual aliquots do not all need to be of the same size.
In another embodiment, the plurality of individual aliquots are spaced at intervals of less than 500 μm, and preferably less than 100 μm. More preferably the spacing is less than 1 μm.
In another aspect, the invention also relates to novel polymers identified as a result of the screening process. Suitable polymers can be formed from one or more of the monomers identified below in the description, examples and Tables. The invention also relates to medical devices containing polymers identified using the process of the invention.
The invention also relates to the use of the process to identify bioadhesive or non-bioadhesive polymers which are suitable for use in medical applications.
The preparation of suitable arrays is described in US2004/0028804 and US2005/0019747 and the contents of these disclosures are specifically intended to form part of the present invention. These documents describe how to make arrays, details of various types of monomers, backing substrates and liquid handling methods.
The early stage biological response to man-made materials is controlled by surface chemistry and topography. In the case of implanted medical devices, this is further influenced by surface conditioning deposition of extracellular tissue fluid components (prior to eventual encapsulation as a foreign body). It is in this period that bacterial adherence occurs. It is not clear why certain polymeric surfaces promote bacterial adherence whilst others do not. Furthermore, there is no empirical way of predicting which polymers will promote adherence. We have found that tailoring the level of adherence provides polymers which have particular advantages in medical applications, relative to existing biopolymers.
For these studies, Pseudomonas aeruginosa PAO1 and Staphylococcus aureus 6390B and uropathogenic Escherichia coli (UPEC) were used as model Gram-negative and Gram-positive bacterial pathogens respectively given their distinct cell envelope structures and surface properties.
The invention thus provides an effective surface-energy based screening method. Polymers exhibiting the required surface energy, measured using the contact angle determination described below, thus will have utility as polymers which are not subject to attack from bacteria or fungus. The invention thus enables identification of materials which may find use in surgery or as implantable devices etc.
The present invention will now be illustrated by means of the following examples of synthetic polymer arrays and methods of assessing microorganism adherence. Included are procedures for preparing the arrays, methods of characterising surface properties and assays for determination of microorganism adherence.
Wetting is the contact between a liquid and a solid surface, resulting from intermolecular interactions when the two are brought together. Wetting is important in the bonding or adherence of two materials. The amount of wetting depends on the energies (or surface tensions) of the interfaces involved such that the total energy is minimized. The degree of wetting is described by the contact angle, the angle at which the liquid-vapor interface meets the solid-liquid interface. If the wetting is very favorable, the contact angle will be low, and the fluid will spread to cover a larger area of the surface. If the wetting is unfavorable, the fluid will form a compact droplet on the surface. Regardless of the amount of wetting, the shape of a drop wetted to a rigid surface is roughly a truncated sphere. A contact angle of 90° or greater generally characterizes a surface as not-wettable, and one less than 90° as wettable. In the context of water, a wettable surface may also be termed hydrophilic and a non-wettable surface hydrophobic. Superhydrophobic surfaces have contact angles greater than 150°, showing almost no contact between the liquid drop and the surface.
In the following Figures,
a) illustrates polar versus dispersive component for all 480 polymers, and Water Contact Angle versus polar component of surface energy for b) polymers containing monomer 10 as their major constituent c) polymers containing monomer 13 as their major constituent d) polymers containing monomer 7 as their major monomer. For figures b) to d) the black star represents the polymer containing 100% of the major monomer, i.e. no minor monomer additions.
Preparation of microarray of polymers: A microarray comprising 480 novel methacrylate/acrylate based polymers was synthesised from 16 major monomers which were mixed pairwise with 6 minor monomers in the following ratios—100:0, 90:10, 85:15, 80:20, 75:25 and 70:30 (
Procedure for contact angle measurements: Contact angles were determined for each polymer on the array prepared according to Example 1 using two liquids: Ultra pure water (18.2 M1 resistivity at 25° C.) and diiodomethane (>99% pure) (Aldrich). A DSA100 (Kruss) with a piezo-doser head was used to dispense a 100 pL droplet of each liquid onto the centre of each polymer spot on the array. Data acquisition was automated with the spot side profile of the back lit spot being recorded. A dual camera system was used, one to record a profile of the spot and the other to record a bird's eye view of the spot to ensure that the water droplet was deposited at the centre of each polymer. Modifications were made to the DSA100 reservoir to allow dosing of liquids with low interfacial tension such as diiodomethane. Data analysis involved following standard contact angle measurement procedures except that due to the small droplet size circle fitting was used instead of Young-Laplace.] 1. Taylor, M.; Urquhart, A. J.; Zelzer, M.; Davies, M. C.; Alexander, M. R., Picolitre water contact angle measurement on polymers. Langmuir Letters 2007, 23, (13), 6875-6878.] Polar and disperse γ values were calculated using the Owens and Wendt's model as described above. Macros were written to enable rapid γ calculations for the large dataset. The surface tension values of the liquids used are provided in Table 1.
Contact angle results and discussion: The automated acquisition and processing of all contact angle data and surface energy calculations were completed within three days. Since this is well within the timeframe required for biological evaluation of such a polymer microarray, this is considered to illustrate the high-throughput nature of the method in this application. Thus, both the water contact angle (WCA) and, upon solution of equation (1), the surface energy (γp & γd) was obtained for all of the 480 novel polymers. The WCA values of the polymers varied greatly from 31° to 104° indicating copolymer surfaces that were hydrophilic to hydrophobic (
The diiodomethane contact angle (DCA) of the polymers varied from ˜13 to 47°. When DCA is plotted against γp it is obvious that there is no relationship between the two parameters (
If γp is plotted against γd it can be observed that the polymers have a narrow range of γd values with a wide range of γp values (
A review of the contact angle data from the copolymer array reveals that monomer structure has a major influence on surface energies. To illustrate this point, three major monomer groups (7, 10 & 13) have been selected in
The addition of minor monomers had a significant effect on the γp of most of the polymers. Minor monomer E generally increases the γp of a polymer (e.g. major monomers 7 & 13) unless the polymer already has a very high γp (e.g. 100% major monomer 10) in which case it will decrease it (
Bacteria and culture conditions: P. aeruginosa (strain PAO1 pUCP18::gfpmut3.1, S. aureus (strain SH1000 pMK4 pXYLA::gfp) and UPEC (pUCP18::gfpmut3.1) were and UPEC routinely grown in Luria-Bertani broth (LB) and Brain Heart Infusion broth (BHI) respectively. All bacterial cells for experimentation were grown to early stationary phase in the specified nutrient both at 37° C. in a 100 ml baffled Erlenmeyer flasks with shaking at 200 rpm unless otherwise stated. Cells were harvested by centrifugation for 10 mins at 4000 rpm (1500 g), washed three times in 25 ml volumes of PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.5 mM KCl, pH 7.2) and resuspended in the working buffer (DPBS, Dulbecco's phosphate buffered saline) by vortex mixing. Bacterial cell suspensions were adjusted to 1.0 (±0.1) by optical density measurements at 600 nm using a UV-VISBLE spectrometer (Elmer Perkin). The spectral characteristics of both GFP-marked bacteria were characterized using a Cary Eclipse Fluorescence spectrophotometer (Varian, UK). All spectra were acquired in 50 mM Tris buffer (1 mM EDTA, pH 7.5).
Combinatorial Array Preparation: Synthetic polymer array chips were prepared according to Example 1 and dried at <50 mTorr for at least 7 days prior to use. The chips were sterilized by exposure to UV for 30 min on each side, and then washed twice with PBS for ≈30 min and then twice with 1×10 ml PBS before bacterial adherence assays, to remove any residual surface contamination.
Hybridization study and microarray scanning: Before hybridization, biomaterial microarrays were pre-wet with 1 mL of 1×PBS for 5 min at room temperature. PBS was removed from the array by shaking and then a 300 μL aliquot of optically adjusted GFP-marked bacterial cell suspension (≈1×108 CFU/ml) was added to the microarray. Cells were evenly distributed on the array surface by covering it with a small piece of parafilm, and the arrays were hybridized for 10 min at room temperature in the dark. After incubation, unbound cells were washed off by delivering 10×1 mL aliquots of 1×PBS over the slide. Slides were allowed to air dry and scanned with a GenePix™ 4000AL four colour fluorescence microarray scanner (Axon Instruments) with an excitation wavelength of 488 nm Fluorescence was detected through a 505-550 nm band pass filter. Images were acquired and processed using the GenePix® Pro microarray image analysis software (version 6.0). The fluorescence images from two arrays are presented in Fig NEW DATA, where one sample has been exposed to P. aeruginosa and the other S aureus.
Table 2 indicates suitable monomers which can also be used in a polymer array according to the present invention.
Previous work in the area has focused on investigating the adherence between mammalian cells and microarrays. We have found that it is possible to provided a microarray containing a range of polymers having a deliberately varied range of surface energies and hence wettabilities in order to probe the adherence of bacteria to the polymer. More importantly, the present invention allows the screening of materials which are resistant to the formation of biofilms when exposed to microorganisms. The adherence or otherwise of bacteria to polymers whilst problematic, is not a significant issue. More importantly, a method of screening for the inhibition of biofilm formation would represent a substantial advantage in a number of medical and non-medical applications. Biofilms are quite different from simple bacterial coatings. In a bacterial coating, individual bacteria act as discrete entities. However in a biofilm the film as a whole is subject to a property known as “quorum sensing” in which individual components which were originally individual bacterial units act in concert and sense one another. Individual units signal via chemical means to one another and effectively act in concert to form a biofilm which has distinct and different properties from the underlying individual components. The biofilm forms as a propagating slimy mass which occludes the surface of a material and which is hard to remove. To date, there has been no method for determining materials upon which biofilm propagation can be inhibited. The present invention provides a method and materials which inhibit biofilm formation on polymeric surfaces. The polymers of the present invention are synthetic polymers. In a preferred embodiment, the polymers are based on acrylate or methacrylate units. Table 3 indicates a preferred group of monomers which can be used in array according to the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0715491.7 | Aug 2007 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2008/050674 | 8/7/2008 | WO | 00 | 4/12/2011 |
