GB2467939A - Removal of bacteria from media - Google Patents

Removal of bacteria from media Download PDF

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Publication number
GB2467939A
GB2467939A GB0902876A GB0902876A GB2467939A GB 2467939 A GB2467939 A GB 2467939A GB 0902876 A GB0902876 A GB 0902876A GB 0902876 A GB0902876 A GB 0902876A GB 2467939 A GB2467939 A GB 2467939A
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bacteria
polymer
binding
media
removal
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GB0902876D0 (en
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Stephen Rimmer
Sheila Macneil
Linda Swanson
Charles William Ian Douglas
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University of Sheffield
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University of Sheffield
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Priority to GB0902876A priority Critical patent/GB2467939A/en
Publication of GB0902876D0 publication Critical patent/GB0902876D0/en
Priority to PCT/GB2010/050294 priority patent/WO2010094976A1/en
Publication of GB2467939A publication Critical patent/GB2467939A/en
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Abstract

A method for the removal of bacteria from media, comprising the steps of: providing a stimulus responsive polymer (e.g. a hyperbranched or highly branched polymer that shows lower critical solution temperature (LCST)) adapted to be capable of binding bacteria; applying said functionalised polymer to the medium so as to facilitate binding of the polymer to bacteria; and removing the polymer and polymer-bound bacteria from the medium. In preferred embodiments the method provides for the use of thermoresponsive polymers functionalised by the incorporation of bacteria-binding ligands in the removal of bacteria from wounds. The invention also envisages the use of the disclosed polymers for the removal of bacteria from media.

Description

NOVEL USE OF POLYMERS
Field of the Invention
[0001] This invention relates to a novel use of polymers for the binding of bacteria.
Specifically, it is concerned with the use of modified stimulus responsive polymers for the binding of bacteria which are found in wounds.
Background to the Invention
[0002] Medical science constantly seeks new developments for the treatment of wounds particularly with a view to reducing the hazards associated with the possible infection of wounds. To this end, there is a constant requirement for the development of new techniques which can facilitate the efficient removal of bacteria from wounds, and it would clearly be desirable if a technique could be developed wherein a wound could be treated with a material which was capable of efficiently binding to the bacteria and thereby facilitating their removal from the wound.
[0003] In WO 2008/059274, the present inventors disclosed various polymers adapted to provide a measurable response associated with a change in the properties of a system. The polymers were typically crosslinked polymers including hydrogels and hyperbranched polymers and their use in the detection of certain biomolecules, such as proteins and bacteria, was proposed. It was suggested that such polymers may thereby find application in biological assays, wherein a measurable change, such as an optical colour change, results from the detection of, for example, bacteria in infected wounds. However, the document did not consider the possible use of polymers for the treatment of wounds or the removal of bacteria from wounds.
[0004] Subsequently, in GB Patent Application No. 0809404.7, the same inventors considered the use of polymers for the separation of cells from samples, and identified polymers which would be suitable for this purpose. Polymers having a temperature-dependent solubility are known as thermoresponsive polymers. For example, polymers such as poly(N-isopropylacrylamide) (PNIPAM or PIPAAm) dissolve in aqueous media below a critical temperature, referred to as the lower critical solution temperature (LOST). As the temperature is raised above the LOST, the polymer forms primary particles which aggregate and then undergo sedimentation in the reaction vessel to form a solid mass.
[0005] PNIPAM and other thermoresponsive polymers had previously been found to adhere to cells above the LOST and, consequently, sheets of thermoresponsive polymer had been used as extracellular matrices on which cells or tissue could be cultured. The polymers were then cooled below the LOST, causing the polymers to dissolve and the cells to be released.
However, since cell adhesion above the LOST is highly sensitive to temperature fluctuations, the use of this technology was limited to environments in which temperature could be controlled within strict limits. In order to address this problem, thermoresponsive polymers had been functionalised with peptides having cell binding properties, these peptides including those having the sequence Arg-GIy-Asp (RGD), which promotes cell adhesion in mammalian tissues through binding to integrins present on cell surlaces.
[0006] Rimmer et a! (Soft Matter, 2007, 3, 971-973) had described the formation of a hyperbranched (highly branched) PNIPAM polymer having chain ends comprising an RGD peptide sequence. It was found that when a solution of this hyperbranched PNIPAM was heated above the LOST, a colloidal dispersion of sub-micron particles, rather than a solid mass of polymer, was formed in the solvent.
[0007] In GB Patent Application No. 0809404.7, the inventors went on to disclose the use of a liquid medium comprising a hyperbranched thermoresponsive polymer for the separation of cells from a sample, the polymer forming a particulate dispersion in the medium above the LCST. The liquid medium was contacted with the sample at a temperature above the LCST of the polymer, such that the polymer was present as a particulate dispersion. The polymer particles subsequently became bound to the cells, allowing the liquid medium to be separated from the sample. 000ling of the liquid medium below the LOST then caused the polymer particles to dissolve and thus release the cells.
[00081 Thus, WO 2008/059274 is concerned with the detection of bacteria by the use of polymers adapted to provide of a measurable response, such as an optical colour change, whilst GB Patent Application No. 0809404.7 relates to the separation of cells from a sample by the use of thermoresponsive polymer. However, neither of these documents considers the use of polymers for the binding of bacteria, thereby facilitating the removal of bacteria from various media, especially from wounds, and it is this issue that the present invention seeks to address, by the provision of polymers adapted to bind to bacteria, and of methods for the removal of bacteria from media such as wounds.
Summary of the Invention
[0009] The present invention, therefore, concerns the use of a stimulus responsive polymer, adapted such that it is capable of binding bacteria, in the removal of bacteria from media, and associated methods for the removal of bacteria. Binding of the bacteria rapidly triggers the collapse of the polymer, such that the collapsed polymer containing bacteria may readily be removed from the media. Typically, the polymer is adapted so that it is capable of binding bacteria by the attachment of suitable ligands capable of binding to bacteria, such as peptides.
[0010] Thus, according to a first aspect of the present invention, there is provided a method for the removal of bacteria from media, said method comprising the steps of: (a) providing a stimulus responsive polymer adapted to be capable of binding bacteria; (b) applying said functionalised polymer to the medium so as to facilitate binding of the polymer to bacteria; and (c) removing the polymer and polymer-bound bacteria from the medium.
[0011] Said stimulus responsive polymer comprises a polymer in which a change is caused to occur by a change in the surrounding environment. Typically, for polymers in solution, the stimuli to which said polymers respond include, for example, changes in temperature, pH, ionic strength, or concentrations of specific materials, such as salts, in the solution. Particularly suitable stimulus responsive polymers in the context of the present invention are thermoresponsive polymers, examples of which include hyperbranched or highly branched polymers that show LOST behaviour in aqueous media. Examples of suitable polymers include, but are not limited to, poly(N-isopropyl acrylamide) and copolymers thereof, highly branched and hyperbranched poly(methyl vinyl ether) and copolymers thereof, and highly branched and hyperbranched poly(vinyl caprolactam) and copolymers thereof.
[0012] Said stimulus responsive polymer is adapted to be capable of binding bacteria by the attachment of suitable ligands which are selected due to their facility to bind to bacteria.
Suitable ligands include various moieties capable of binding to bacteria, and these may include groups derived from peptide antibiotics and their bacteria binding derivatives, non-peptide antibiotics and their bacteria binding derivatives, antibody fragments that bind to bacteria, and oligo-and mono-saccharides that bind to bacteria. Other examples of suitable ligands include any low molecular weight compounds that selectively bind to any cell surface receptor on bacteria. Especially suitable ligands for present purposes are bacteria-binding peptides, which may include antibiotic and non-antibiotic peptides.
[0013] Application of said stimulus responsive polymer to a bacteria-containing medium may be conveniently achieved by the application of the functionalised polymer in a solution or tethered to a surface or gel. In both variants, the polymer is below the LOST (in the open coil state) in the absence of bacteria. The interaction with bacteria drives the polymer into its collapsed state and, in this state, polymer plus bacteria can be removed. If subsequently cooled the polymer changes back to its open coil state and releases the bacteria.
[0014] It is necessary that said polymer is applied to the medium at a temperature below the lower critical solution temperature (LOST) of the functionalised polymer, which is typically above 60°C. Consequently, there is no requirement for precise control of temperature in the handling of this polymer because it will not spontaneously change conformation at room temperature or body temperature, but will only collapse when bacteria bind to it. Removal of the polymer from the medium then allows for removal of the polymer-bound bacteria. The method of the invention finds particular application when the medium is a wound, i.e. in the removal of bacteria from wounds.
[0015] According to a second aspect, the present invention provides for the use of a stimulus responsive polymer adapted to be capable of binding bacteria in a method for the removal of bacteria from media. Preferred media comprise wounds.
Brief Description of the Drawings
[0016] A better understanding of the invention will be facilitated by reference to the accompanying illustration, wherein: Figure 1 provides a comparison between (A) the prior art application of polymers for the lifting of mammalian cells from cell culture surfaces, and (B) the method of the present invention for the removal of bacteria from wounds.
Figure 2 provides an illustration of how a stimulus responsive polymer is insolubilised on incubation with bacteria according to the method of the invention.
Figure 3 shows the visible removal of bacteria from an infected skin wound model when treated with stimulus responsive polymers according to the method of the invention; and Figure 4 provides a quantitative graphical illustration of the removal of bacteria from an infected skin wound model according to the method of the invention.
Detailed Description of the Invention
[0017] In a preferred embodiment, the invention provides a method for the removal of bacteria from wounds which involves the use of a stimulus responsive polymer to which antibiotic peptides have been attached, such that these polymers are capable of binding bacteria.
Application of the polymer to the wound is carried our below the LOST of the polymer, such that the polymer is in open chain form. The subsequent binding of the bacteria to the polymer then rapidly triggers the collapse of the polymer chain, whereupon the collapsed polymer containing bacteria can then be removed.
[0018] The claimed method finds particular application in the treatment of wounds, for reducing the bacterial content of chronic infected wounds. However, the method can also be used for many other applications, including the removal of bacteria from liquids that are passed over the polymer in, for example, microfluidic devices, or wherein the polymer is adhered to a catheter. The key feature of the invention is in the ability of the polymer to physically bind to bacteria, and this is particularly useful in the reduction of bacterial contamination in an infected wound. The method of the invention is also of use in the decontamination of medical devices.
For example, natural and synthetic polymer scaffolds for use in tissue engineering can be decontaminated by application of a bacteria binding polymer providing a mild route to disinfection and sterilisation.
[0019] As previously noted, preferred stimulus responsive polymers that show changes in response to changes in the environment in the context of the present invention include polymers of N-isopropyl acrylamide (NIPAM). The macroscopic changes that can occur at critical values of pH, temperature or ionic strength with such polymers come about because the polymer changes from an open (solvated) coil to a collapsed state known as a globule. Any aqueous polymer solution will respond in this way to changes in pH, temperature or ionic strength but, for many systems, the critical points occur at temperatures that are above 100°C or below 0°C. However, by the introduction of specific features on either the ends of polymer chains or on crosslinked (hydrogel) chains, the temperature of the transition of a thermoresponsive polymer can be reduced. Thus, if a polymer is observed at a particular temperature that is below the non-bound transition temperature, but above the bound transition temperature, a coil-to-globule transition will be observed on binding.
[0020] In the case of poly(NIPAM) materials, as disclosed in GB Patent Application No. 0809404.7, cell adhesion to the polymer is also enhanced above the transition temperature, whereas below the transition temperature cell adhesion is not supported. The cell adhesive state can be enhanced by the addition of cell adhesive peptides and, thereby, particulate versions of these peptide functional PNIPAM materials can be used to lift cells from cell culture surfaces.
[0021] In the case of the present invention, it is seen that poly(NIPAM) materials may be functionalised with peptides that bind to bacteria, thereby facilitating the removal of bacteria from media such as wound beds. Preferred bacteria binding peptides are based on antibiotics, but non-antibiotic species that bind bacteria also function effectively.
[0022] Referring now to the illustrations provided in Figure 1, there is initially shown, in section (A) a process for the lifting of mammalian cells from cell culture surfaces, wherein mammalian cells are exposed at a temperature above the transition temperature (10-25°C) to polymer particles functionalised with a cell adhesive peptide. The cells are then lifted from the plate and remain suspended, after which the suspension is transferred and cooled.
[0023] In section (B), on the other hand, there is seen a polymer functionalised with a bacteria-binding-ligand, which is added to a wound below the transition temperature, such that the polymer is in the open chain form. It subsequently binds to the bacteria in the wound and, on so binding, the polymer chain collapses. In this collapsed state, bacteria adhere to the polymer rather than the wound and can thus be physically removed from the wound bed as a bacteria-polymer aggregate.
[0024] Removal of the polymer, with bacteria bound thereto, may be achieved simply by maintaining the polymer at room temperature or above. Thus, removal of bacteria is typically achieved by rinsing the wound with warm sterile saline or swabs soaked in warm saline solution will achieve removal of the polymer with bacteria firmly attached. If the system is then deliberately cooled to a point below the bound transition temperature (for example, a temperature below 10°C), then release of the bacteria from the polymer is observed. This observation is explained by the fact that the method of the invention involves not only the binding of bacteria to the polymer, but also the lowering of the LOST once the polymer becomes so bound. In polymers which are appropriate for use in the disclosed applications, the associated decrease in LOST takes the LOST below the application temperature, such that the polymer open coil chain collapses into the globule on binding. Both chain collapse and binding are required in order for removal of bacteria to occur so that, if the temperature is artificially decreased to below the bound LOST, then the polymer reverts to the open coil state, with the consequence that the bacteria are released.
[0025] Looking at Figure 2, there can be seen the effect on a stimulus responsive polymer of incubation with bacteria, wherein the polymer becomes insoluble only after such incubation.
Thus, in image a) is illustrated a gross view of S.aureus bacteria in buffer solution, whilst image b) shows the effect of fluorescence microscopy on fluorescently labelled (fluorescein) S.aureus in buffer solution, wherein bacteria are in suspension, and spread evenly throughout. Image c) is a gross view of a control polymer, which is non-stimulus responsive, in buffer solution alone, and it is seen that this control polymer becomes insoluble even without the presence of bacteria. From the fluorescence microscopy of fluorescently labelled S.aureus incubated with control polymer as shown in image d), as with image b) it may be gleaned that the bacteria remain evenly distributed throughout. Image e) presents a gross view of the stimulus responsive polymer BPNIPAM-VAN in buffer solution alone. Unlike the control polymer, the functionalised polymer remains in solution. Image f) is obtained by fluorescence microscopy of BPNIPAM-VAN in buffer solution alone and, as expected, there no fluorescence. Image g) shows a gross view of BPNIPAM-VAN incubated with S.aureus, from which it may be seen that binding of bacteria appears to render the polymer insoluble, and causes it to form visible aggregates, whilst the fluorescence microscopy image h) of BPNIPAM-VAN incubated with fluorescently labelled S.aureus indicates that the fluorescent bacteria form aggregates as they are bound to the collapsed polymer.
[0026] Figure 3 illustrates the visible removal of bacteria from an infected wound model by the application of a stimulus responsive polymer. Thus, tissue engineered skin constructs (a) are burnt (b) so as to create a burn-wound model and allow bacteria entry to the skin, as occurs in vivo. Wounded skin thus obtained was infected with the common pathogenic bacteria Pseudomonas aeruginosa for 24 hours, after which time either the polymer, or buffer alone as control, were applied to the surface of the skin. After one hour, the polymer or buffer was removed, and the skin was examined using histology to visualise the level of infection. In 1 5 infected skin sections incubated with buffer alone, a large number of bacteria were visible within the skin, particularly along the surface, and this is illustrated in image (c) in the depicted layer whereas, in infected skin sections incubated with polymer, the majority of visible bacteria are clearly no longer present within the skin sections as illustrated in image (d). Clearly, therefore, it appears that bacteria become bound to the polymer and are removed upon removal of the polymer.
[0027] A quantitative illustration of the removal of bacteria from an infected wound model by the application of a stimulus responsive polymer is shown in Figure 4, wherein the numbers of viable bacteria remaining in the tissue from engineered skin constructs burnt and infected as in Figure 4 were quantified. Thus from graph a) it is seen that, in skin samples incubated with polymer, the numbers of bacteria remaining in the tissue following removal of polymer are substantially reduced as compared to those incubated with buffer alone whereas, conversely, graph b) shows that the numbers of bacteria found in the polymer solution removed from the wound are substantially increased as compared to those found in buffer alone.
[0028] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
[0029] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
[0030] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Claims (19)

  1. CLAIMS1. A method for the removal of bacteria from media, said method comprising the steps of: (a) providing a stimulus responsive polymer adapted to be capable of binding bacteria; (b) applying said functionalised polymer to the medium so as to facilitate binding of the polymer to bacteria; and (c) removing the polymer and polymer-bound bacteria from the medium.
  2. 2. A method as claimed in claim 1 wherein said stimulus responsive polymer comprises a polymer in solution in which a change is caused to occur by a change in the temperature, pH or ionic strength of the solution.
  3. 3. A method as claimed in claim 1 or 2 wherein said stimulus responsive polymer comprises a thermoresponsive polymer.
  4. 4. A method as claimed in claim 3 wherein said thermoresponsive polymer comprises a hyperbranched or highly branched polymer that shows LOST behaviour in aqueous media.
  5. 5. A method as claimed in claim 4 wherein said polymer is selected from poly(N-isopropyl acrylamide) (NIPAM) and copolymers thereof, highly branched and hyperbranched poly(methyl vinyl ether) and copolymers thereof, and highly branched and hyperbranched poly(vinyl caprolactam) and copolymers thereof.
  6. 6. A method as claimed in any one of claims 1 to 5 wherein said stimulus responsive polymer is adapted to be capable of binding bacteria by the attachment of ligands possess the facility to bind to bacteria.
  7. 7. A method as claimed in claim 6 wherein said ligands comprise bacteria-binding moieties.
  8. 8. A method as claimed in claim 7 wherein said bacteria-binding moieties comprise groups derived from peptide antibiotics and their bacteria binding derivatives, non-peptide antibiotics and their bacteria binding derivatives, antibody fragments that bind to bacteria, and oligo-and mono-saccharides that bind to bacteria.
  9. 9. A method as claimed in claim 7 wherein said bacteria-binding moieties comprise antibiotic or non-antibiotic peptides.
  10. 10. A method as claimed in any one of claims 1 to 9 wherein application of said stimulus responsive polymer to a bacteria-containing medium is achieved by the application of the functionalised polymer in a solution or tethered to a surface or gel wherein the polymer is below the lower critical solution temperature (LOST) of the functionalised polymer in the absence of bacteria.
  11. 11. A method as claimed in any preceding claim wherein the medium is a wound.
  12. 12. A method as claimed in any one of claims 1 to 10 wherein the medium comprises a liquid.
  13. 13. A method as claimed in claim 12 wherein the liquid is passed over the polymer in a microfluidic device or the polymer is adhered to a catheter.
  14. 14. The use of a stimulus responsive polymer adapted to be capable of binding bacteria in a method for the removal of bacteria from media.
  15. 15. The use as claimed in claim 14 wherein said media comprise wounds.
  16. 16. The use as claimed in claim 14 wherein said media comprise liquids.
  17. 17. The use as claimed in claim 14 wherein said media comprise medical devices.
  18. 18. A method as hereinbefore described and with reference to the accompanyingdescription and drawings.
  19. 19. The use as hereinbefore described and with reference to the accompanyingdescription and drawings.
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