Method and apparatus for producing articles from bacteriophage-containing filaments

Abstract

The present invention provides a method for manufacturing an article 10 including one or more filaments 12 of a material having a hydrophilic surface 12a, the method comprising the steps of forming a first solution 16 containing one or more bacteriophages 18a suspended in a carrier fluid 20, depositing the first solution 16 onto one or more filaments 12 by dispensing droplets 22 thereof from a droplet dispenser 24 to deposit a predetermined amount of the first solution per unit length onto the first filaments 12, and then forming the one or more first filaments 12 into a final product 10.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to methods of producing articles having bacteriophage attached thereto, and particularly, but not exclusively, to methods of producing articles comprising or formed of filamentous material having bacteriophage deposited thereon. The invention also relates to articles produced according to the methods, and to articles comprising one or more filaments of material containing bacteriophage thereon or therein. Other elements of the invention include apparatus for producing articles, articles produced by the methods described herein, and apparatus and methods for arraying bacteriophage on filaments.

[0002] 2. Background of the Invention Resistance to traditional antibiotics is becoming increasingly prevalent and threatens to become the next greatest challenge to human health, predicted to result in 10 million deaths per year and increased costs to healthcare systems after 2050. One promising alternative method to prevent and treat bacterial infections is the use of bacteriophages, natural viruses and enemies of bacteria.

[0003] One of the challenges of bacteriophage therapy is the instability of the bacteriophages when stored in solution and their rapid clearance from the human body when administered as an alternative treatment to antibiotics. The particular instability of the bacteriophage cocktail was highlighted in a paper published in 2015 summarizing the first and largest European clinical trial of bacteriophage therapy.

[0004] It is known to stabilize bacteriophages by covalent attachment onto a substrate material, as described in the original patent EP 1 496 919, which may involve covalently attaching the bacteriophage to a surface by corona activating the surface or by using a coupling agent. It is also known to protect bacteriophages during storage by treating them with glutaraldehyde of trehalose.

[0005] Although the patent claims to allow for specific binding of the head groups, the methods described only allow for random binding of bacteriophages to surfaces and do not prevent horizontal alignment of the tails to the surface or suggest a way to avoid horizontal alignment of the tails to the surface. Bacteriophages bind to bacteria through their tails, and horizontal alignment of bacteriophages on surfaces may reduce the infectivity of the bacteriophages and therefore their effectiveness against pathogens.

[0006] The patent also claims that multiple strain-specific bacteriophages can be attached to a surface, but the method only allows for random addition of a solution containing a mixture of different bacteriophage strains. Unfortunately, this approach may not be as effective as desired, as it is well known that cocktails of bacteriophages in solution can interfere with each other, and complete addition may reduce overall infectivity.

[0007] Another patent, entitled "Production of Immobilized Bacteriophage" (EP 18207889), teaches pressure spraying microdroplets containing bacteriophage onto microparticles or filaments or planar surfaces. Disadvantages of this method include the inability to deposit precise concentrations of bacteriophage onto each particle / filament, as pressure spraying results in random spreading of the microdroplets onto the powder, and the inability to efficiently deposit high concentrations of bacteriophage onto each particle, as the volumes involved are small.

[0008] While this second patent suggests a more even distribution of bacteriophage onto the particles, it does not guarantee that every particle or substrate receives a very precise concentration of bacteriophage, nor does it guarantee that no bacteriophage will be wasted by missing the target substrate when distributed over a surface.

[0009] In this second patent, an electric field is applied to both the material and the solution containing the bacteriophage, promoting the attachment of the phage capsid to the material surface. However, after the bacteriophage are deposited, the surface of the material is not negatively charged to attract the positively charged tails to an optimal position. Thus, not all bacteriophages have their active tails pointing outwards from the attachment surface.

[0010] Other methods described in the literature involve immersing a material in a solution containing bacteriophage and coating the material with the bacteriophage present in the solution, however this method does not ensure the transfer of precise amounts of bacteriophage between the solution and the material, and results in random deposition of bacteriophage in random areas of the material, resulting in a high proportion of bacteriophage being wasted in the solution.

[0011] In the manufacture of products that use bacteriophages, the cost of the bacteriophages used to manufacture the product accounts for a large portion of the product's cost. Poorly aligned bacteriophages reduce the effectiveness of the bacteriophages against pathogens that require more bacteriophage. Existing manufacturing methods have poor control over bacteriophage concentration and usage, resulting in waste of expensive bacteriophage solution.

[0012] It is an object of the present invention to avoid and / or mitigate at least some of the above mentioned disadvantages. A more specific object of the present invention is to provide an alternative method for producing articles consisting of or formed of filaments of material containing bacteriophage, where for the first time a very precise concentration of bacteriophage can be precisely immobilized on a defined surface area of ​​a substrate, reducing and possibly eliminating waste of bacteriophage solution and allowing the application of patterns of multiple bacteriophage strains on the same or different zones and / or areas of the article being formed. This also has the advantage of allowing the addition of multiple cocktails of synergistic phages against the same bacterial strain in the same device, while keeping antagonistic phages separate, or allowing the addition of multiple cocktails targeting different bacteria. Other objects of the present invention include, but are not limited to, providing an apparatus suitable for use in the method of the present invention and an article produced by the method of the present invention. Yet another aspect of the present invention provides a method and apparatus for aligning bacteriophage with respect to a filament to ensure that the tails of the bacteriophage are aligned away from the filament. The invention also allows for the production of filaments that can be stored for later assembly with other filaments containing the same or different bacteriophages into finished articles. Desirably, such articles can be custom-made with a specific bacteriophage selected to suit the particular medical application of the article. Thus, the invention allows for the production of off-the-shelf designer or custom-made products.

[0013] The experiments contained herein show that excess bacteriophage solutions deposited in the same or overlapping locations can reduce the efficacy of the finished product, even if the solutions contain the same bacteriophage. The reduction in efficacy can be even worse if the droplets are solutions containing different bacteriophages that have antagonistic effects.

[0014] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT According to one aspect of the invention, there is provided a method for manufacturing an article comprising one or more filaments of a material having a hydrophilic surface, the method comprising the steps of forming a first solution containing one or more bacteriophages suspended in a carrier fluid; providing one or more first filaments; depositing the first solution containing the one or more bacteriophages onto the one or more first filaments, characterized in that the deposition of the first solution is performed by dispensing droplets thereof from a droplet dispenser to deposit a predetermined amount of the first solution per unit length onto the first filaments; and forming the one or more first filaments into a final article.

[0015] The method may include forming a second solution containing one or more second bacteriophages different from the first bacteriophage suspended in a second carrier fluid; providing one or more second filaments; depositing the second solution containing the one or more second bacteriophages onto the one or more second filaments, wherein a predetermined amount of the second solution per unit length is deposited on the second filaments; and forming the one or more first filaments together with the second filaments into a final article.

[0016] Optionally, one or more first filaments each have a first region distinct from a second region. The method optionally further includes forming a second solution containing one or more second bacteriophages different from the first bacteriophage suspended in a second carrier fluid, depositing a predetermined amount of the first bacteriophage solution per unit length onto the first regions, depositing a predetermined amount of the second bacteriophage solution per unit length onto the second regions, and forming the one or more filaments (12) into a final article (10).

[0017] The forming step may include one or more of weaving, braiding, plating, knitting, embroidering the article, or forming a nonwoven article.

[0018] Preferably, the bacteriophage is 10 2 PFU / mL ~ 10 12 The bacteriophage is mixed into the solution at a concentration of between 10 PFU / mL of the solution. Even more preferably, the bacteriophage is mixed into the solution at a concentration of between 10 5 PFU / mL ~ 10 9 The bacteriophage are mixed into the solution at a concentration of between 100 and 200 PFU / mL of solution.

[0019] Advantageously, the filament has a length L and the method includes depositing the solution at discrete spaced locations along the length L of the filament.

[0020] The method may include simultaneously depositing multiple droplets of the solution onto the filament at distinct, spaced apart locations along the filament, preferably with each of the multiple droplets at distinct, spaced apart locations along the filament, allowing for precise saturation of the filament and / or separation of different phage solutions.

[0021] The method may include depositing the droplets on the filament at intervals sufficient to saturate the entire length L of the filament or substantially the entire length of the filament.

[0022] The method may include depositing the droplets onto the filament at intervals insufficient to saturate the entire length of the filament, thereby forming a discrete length of the filament to which no bacteriophage is applied.

[0023] For example, by using a dead solution, it can be easily shown experimentally that a droplet of a certain volume V will saturate a filament of a certain diameter over a length S, and the length of the saturated filament can be emphasized and measured.

[0024] The method includes manufacturing an article having two or more zones and forming the article from one or more filaments having different bacteriophages, thereby forming an article having different bacteriophages in its different zones.

[0025] The article may have an end and a non-end, and the method may further include forming the article with a first filament having a first bacteriophage at the end and a second filament having a second bacteriophage at the non-end.

[0026] The method may include mixing a solution containing the bacteriophage prior to deposition onto the filament.

[0027] The method may also include modifying a surface of the filament to make it more hydrophilic. Modifying the surface of the filament may be performed by passing the filament through a plasma discharge.

[0028] A particularly advantageous arrangement involves passing the filament along an electrode and applying a potential to align the bacteriophage with respect to the filament. This may be done by passing the filament through a tubular electrode. Such electrodes may be cylindrical, square, triangular in cross section, or any other shape, so long as they are capable of generating an electric field potential that will align the bacteriophage as described herein. It is also possible to pass the filament between spaced apart electrodes.

[0029] In one arrangement, a first solution is deposited by simultaneously dispensing multiple droplets thereof from spaced droplet dispensers onto the filament.

[0030] According to a further aspect of the invention, there is provided a method for manufacturing a filament, comprising the steps of forming a first solution containing one or more bacteriophages suspended in a carrier fluid, providing one or more filaments, and depositing the first solution containing the one or more bacteriophages onto the one or more first filaments, wherein the depositing of the first solution is performed by dispensing droplets thereof from a droplet dispenser to deposit a predetermined amount of the first solution per unit length onto the first filaments.

[0031] The method of the second embodiment comprises: 2 PFU / mL ~ 10 12 The concentration of bacteriophage per unit solution is between 10 PFU / mL or 10 5 PFU / mL ~ 10 9 The method may include mixing the bacteriophage in the solution at a preferred concentration of bacteriophage per unit solution of between PFU / mL.

[0032] The filament may have a length L, and the method may include depositing the solution at discrete spaced locations along the length L of the filament.

[0033] The method may include simultaneously depositing a plurality of droplets of the solution onto the filament at distinct spaced locations along the filament.

[0034] The method may also include depositing the droplets onto the filament at intervals sufficient to saturate the entire length of the filament, L. Alternatively, the method may include depositing the droplets onto the filament at intervals insufficient to saturate the entire length of the filament, L, thereby forming a separate length of the filament to which no bacteriophage has been applied.

[0035] Advantageously, the method includes a step of mixing a solution containing said bacteriophage before depositing it on said filament.

[0036] For some materials, a step of modifying the surface of the filament may be included to increase the hydrophilicity of the surface. Modifying the surface of the filament may be performed by passing the filament through a plasma discharge.

[0037] A particularly advantageous arrangement involves passing the filament along an electrode and applying a potential to align the bacteriophage with respect to the filament. This may be done by passing the filament through a tubular electrode. Such electrodes may be cylindrical, square, triangular or any other shape in cross section, so long as they are capable of generating an electric field potential that will align the bacteriophage, as described herein. It may also be possible to pass the filament between spaced apart electrodes.

[0038] Preferably, the droplets are dispensed having a size greater than 0.1 μL, more preferably, the droplets are dispensed having a size range between 0.2 μL and 100 μL.

[0039] According to yet another aspect of the present invention, there is provided an article comprising one or more filaments of a material having a hydrophilic surface and containing one or more bacteriophages within said hydrophilic surface.

[0040] The invention also describes a method to use charges around the material after the bacteriophage have been deposited to ensure that the bacteriophage tails are oriented perpendicular to the substrate surface, enhancing infectivity.

[0041] Aspects of the invention will now be more particularly described, by way of example, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0042] [Figure 1] FIG. 1 is a schematic diagram of a woven fabric structure having filaments incorporating the present invention. [Diagram 2]2a-2c are examples of alternative braiding structures for filaments to which the present invention may be applied. [Diagram 3] 3a-3c are further examples of woven, twisted and / or braided structures to which the present invention may be applied. [Figure 4] FIG. 4 is an example of a nonwoven structure to which the present invention can be applied, the nonwoven structure comprising a wadding of randomly distributed filaments to which the method of the present invention can be applied. [Diagram 5] FIG. 5 is a schematic diagram of a first droplet dispensing arrangement. [Figure 6] FIG. 6 is a schematic diagram of an alternative droplet dispensing arrangement. [Figure 7] FIG. 7 shows the filament after receiving multiple droplets. [Figure 8] FIG. 8 is a schematic diagram of an apparatus arrangement suitable for use in producing filaments according to the present invention. [Figure 9] FIG. 9 is a close-up view of the configuration used to array bacteriophage on the filament. [Figure 10] FIG. 10 shows a sample of a final product that may be made from filaments subjected to the method of the present invention. [Figure 11] FIG. 11 illustrates several exemplary configurations of bacteriophage combinations applied to multiple regions of one or more filaments. [Figure 12] FIG. 12 shows four groups of three silk filament samples each having four microliter drops of the same S. aureus phage solution applied to them. [Figure 13] FIG. 13 shows six separate droplets of 4 microliters of phage solution applied to six groups of three silk filament samples, respectively.

[0043] Referring now to the drawings generally, and more particularly to Figures 1-4, an article or product 10 of the present invention comprises one or more filaments 12 of a material having a hydrophilic surface 12a, which may be natural to the material or may be produced in accordance with one element of the present invention. The article 10 may take any one of a number of forms produced by intercrossing one or more filaments 12a, 12b, 12c, 12d (for example). The intercrossing may be by weaving as shown in Figure 1, plating or knitting as shown in Figures 2a-2c, twisting / plating as shown in Figures 3a-3c, the filaments 12 may be embroidered into a material or fabric, or may comprise a wadding (non-woven form) of randomly distributed filaments as shown diagrammatically in Figure 4. Such filaments are useful in the manufacture of articles such as surgical sutures, artificial ligaments, tendons, devices for knee or hip implants, vascular grafts, nerve repair conduits, ties, wadding for wound dressings, protective coverings, fabrics for hernia repair or breast implants.

[0044] The filaments may be of natural materials such as silk, cotton, wool, etc., or of artificial materials such as polyester, polyethylene, polypropylene, polytetrafluoroethylene, prolene, nylon, polydioxanone, etc. The surfaces of some of these materials may already be hydrophilic, as in the case of silk, cotton, and some wool, but artificial materials may not necessarily have a naturally hydrophilic surface and may need to be treated or modified to make them suitable for use in the present arrangement. Details of one method for making the surface of an artificial material hydrophilic are alternatively described herein, but it is understood that other methods of achieving a hydrophilic surface may be used. Some such alternatives include roughening the surface with an abrasive, acid etching, electron bombardment, etc. Any of these methods effectively modify the surface of the material forming the filament to increase its hydrophilicity.

[0045] The process of attaching bacteriophage to the filament begins with the preparation of a solution 16 containing one or more bacteriophage 18 held in suspension in a carrier fluid. The carrier fluid may comprise phosphate buffered saline (PBS), or injection broth, or SM buffer, or ethanol, although other carrier fluids may be used. The carrier fluid is important because it allows the bacteriophage to be suspended in something that can be applied to the filament, perhaps allowing for better control of application and more certainty of bacteriophage distribution than has been possible with conventional processes. There is a wide range of concentrations at which bacteriophage can be used in suspension, but a good concentration suitable for use in the present arrangement is between 10 and 200 μg / ml. 2 PFU / mL ~ 10 12 It has been found that the bacteriophage concentration per unit solution in PFU / mL is 10 5 PFU / mL ~ 10 9 PFU / mL because this range ensures that sufficient bacteriophage are present, but avoids overloading the solution.

[0046] Once the solution 12 is prepared, it may be dispensed in any one of a number of ways, but it is preferred that the dispensing technique is one of droplet dispensing, as this has been found to provide a high degree of control over the delivery process and the density of the bacteriophage concentration on the finished filament and final article. A distinction is made between dripping and spray coating techniques, the latter being well known for use as a mechanism for applying bacteriophage to articles. Spray coating techniques can dispense multiple small or microdroplets, as opposed to droplet dispensers that can dispense droplets individually at a known and controlled rate. Spray techniques, while suitable for some surfaces, such as substantially flat surfaces, tend to produce undesirable waste from overspray and can cause washing away of the desired bacteriophage as the carrier solution drains from the surface to which it has been applied. In such an arrangement, it is difficult to ensure a good and / or accurate concentration of the bacteriophage, and to coat the bacteriophage evenly across the entire surface to which it has been applied. While this may be acceptable in some applications, it is generally compensated for by applying a much larger amount / concentration of bacteriophage than would be considered desirable or even economically desirable. This problem is what one element of the present invention aims to solve. Droplets tend to have a size range between 0.005 microliters and 0.08 microliters, whereas droplets have a size range between 0.2 microliters and 100 microliters. In some applications of the present invention, advances in single microdroplet dispensing technology allow for smaller discrete droplet sizes, now down to 0.1 microliters. Importantly, droplets can be dispensed and dispensed one drop at a time or as discrete droplets by a droplet dispenser, allowing the droplets to be placed at selected discrete locations. The present invention takes advantage of the inherent properties of droplets to control the application of bacteriophage in a manner that is in stark contrast to prior art approaches, and that is measured, reproducible, precise, and economical, which is important for quality control.These and other advantages of the present invention are described in more detail later in this specification.

[0047] FIG. 5 is a schematic diagram of a droplet dispensing apparatus 1, which may be used in conjunction with other aspects of the invention, to precisely dispense and deposit droplets of carrier fluid 20 (having bacteriophage 18 in suspension). The apparatus may include one or more reservoirs 40 of premixed bacteriophage solution 16, containing carrier fluid 20 with a known or predetermined concentration of bacteriophage 18. A mixing apparatus, shown generally at 42 in FIG. 5, including a drive mechanism 44 and mixing paddles 45, may be provided integral with one or more of the reservoirs 40 to keep the bacteriophage 18 and carrier fluid 20 circulating within the one or more reservoirs 40, thereby ensuring a more uniform distribution of the bacteriophage 18 within the carrier fluid 20. However, it will be appreciated that such a mechanism is not necessary in all applications, and may not be necessary where the solution 16 is to be used within a short period of time after being placed in the reservoirs 40. Also, one or more of the multiple reservoirs 40 are provided with a droplet dispenser 46, which may include a solenoid 48, piezoelectric actuator 48 or other actuation device 48 acting on a flow control means 49, such as a valve 49, to control the timing of droplets dispensed therefrom. A timing controller, shown diagrammatically at 50, may be coupled to each solenoid 48 to govern the timing and / or duration of any droplets that may be dispensed. Also, if necessary, an indexing / advancing system (not shown, but represented by arrows 52) may be used to move the filament 12 under one or more dispensers 46 to allow droplets 54 to be dispensed onto the filament 12, as will be described in more detail later in this specification. FIG. 5 shows an arrangement with multiple reservoirs 40, each containing the same solution 16 and represented by the letter A. If the device is used to dispense the same solution 16, the separate reservoirs 40 may be replaced by a common reservoir. FIG. 6 shows a slight variation of the illustrated arrangement, which differs entirely by the presence of multiple reservoirs 40, each containing a different solution 16.This configuration can be used to dispense different combinations of bacteriophage solutions A, B, C, D onto the same filament 12, but at different locations thereon. The configurations of Figures 5 and 6 may be used to apply different cocktails or combinations of bacteriophages 18 to different filaments, or to apply the same bacteriophage to multiple filaments. Furthermore, they may be used to apply different bacteriophages 18 to different regions R on the same filament. In fact, the ability to space the regions R to which the bacteriophages 18 are applied solves another problem, since it is well known that different bacteriophages 18 or cocktails thereof, when combined together at one location, may negatively interact with each other, thereby reducing rather than enhancing the effectiveness of the bacteriophage solution 16.

[0048] FIG. 7 shows a filament 12 passed through the apparatus of FIG. 5 or FIG. 6, showing dispensed droplets 100 spreading laterally as indicated by arrows S within the hydrophilic surface (12a) to distribute the solution 16 and bacteriophage 18 along the length of the filament 12. The duration Dd of the droplets, and therefore the volume V, or the distance traveled by the advancement system 52, can be controlled by the controller 56 to control the length of the filament 12 saturated by each droplet and the distance between each droplet, respectively, to cause complete coating of the filament or to leave gaps G between the bacteriophage-impregnated portions W, X, Y, Z. Spaced regions R1, R2, etc. of the bacteriophage-coated length can be utilized to reduce the amount of solution applied while still ensuring sufficient space or volume available for adsorption of the solution 16, thereby reducing or even eliminating the possibility of waste.

[0049] It can be easily established by experiment that for a certain size filament 12, the distance S is saturated by the volume V of bacteriophage solution 18. The indexing / filament advancement system 52 of the droplet dispenser 1 is configured to move the filament 12 at least the distance S between the dispensing of each droplet of the volume V to provide a saturated filament free of waste bacteriophage solution. The indexing / filament advancement system 52 of the droplet dispenser 1 may be configured to move the filament 12 more than the distance S between the dispensing of each droplet of the volume V to provide a filament having a gap G between the saturated regions R. Those skilled in the art will appreciate that a given volume V may be provided by multiple droplets at each distinct location. A filament 12 having a gap G between the saturated regions R may be particularly useful when multiple solutions 18 containing different bacteriophages or different cocktails of bacteriophages are applied to the same filament 12. The filament advancement system 52 may stop the filament 12 as each droplet is dispensed, or the filament 12 may be moved continuously by the filament advancement system 52 and the dispensing of droplets by the droplet dispenser 46 may be indexed relative to the movement of the filament 12 by the filament index / advancement system 52.

[0050] FIG. 11 shows several exemplary configurations of bacteriophage 18 combinations applied to multiple regions R1-R4 of one or more filaments 12 to attack one or more bacteria with multiple phages 18, thereby increasing efficacy.

[0051] FIG. 8 is a schematic diagram of four components of an overall apparatus that can be used to pre-treat the filament 12, apply droplets 54, and also post-treat the filament to align the bacteriophage 18 in a manner that will be described in more detail below. Each component can be used separately or in combination with any one or more of the other components, but preferably they are all used together. In either arrangement, the first component includes an optional bath 60, for example of hydrogen chloride, that is used to clean the surface of the filament and pre-treat the surface to make it more susceptible to adsorption of a subsequently applied treatment. The second optional component includes a plasma generator 64 that surrounds the filament 12 and can generate a plasma, such as a corona plasma, for the purpose of increasing the hydrophilicity of the surface, a well-known technique in the art and often referred to as "surface modification" that alters the chemistry of the filament surface. In essence, the plasma reacts with the surface 12a of the filament 12, making it more porous and therefore more susceptible to adsorption of fluids. This process is suitable for use with artificial fibers as mentioned above, but is less commonly used with natural fibers such as cotton, silk, etc., each of which is already relatively hydrophilic. The droplet dispensing mechanism of Figures 5 and 6 is located after the optional plasma generator 64 and includes the components described above with reference to Figures 5 and 6 and will not be described again at this point. The last optional component includes an electric field generator 66, formed for example of a tubular electrode 68 having a longitudinal axis X, through which the filament 12 passes so as to be exposed to any electric field generated therein. An electric potential EP is generated between the filament and the electrode 68 such that the electrode is negative and the filament is positive relative to the electric field. The generation of this electric potential has the effect of aligning each bacteriophage radially outward of the filament, as shown diagrammatically in Figure 9. The positive tail 18b of the bacteriophage 18 is attracted to the electrode 68 and the negatively charged head 18a is attracted to the filament 12. This orientation is maintained after the filaments and bacteriophage 18 have passed through the electric field generator 66 and will remain in this orientation for a significant amount of time and / or unless realigned by some other external influence.This alignment is important because it is the tail of the bacteriophage that attacks and destroys bacteria, and orienting the bacteriophage in this way makes them more effective at destroying bacterial infections adjacent to the location where they are used.

[0052] FIG. 10 shows a preferred example of a complete medical product 120 that may be formed from multiple filaments coated in the manner described above. The example is an artificial ligament formed from a woven / braided reband of material 122 folded to form soft loop ends 124 and stiff loop ends 126. The filaments 12 may also be delivered to the patient within a capsule 130, a pill 132 containing the filaments 12, or a fibrous capsule 134. Other forms of ligaments, delivery systems, and indeed other forms of medical products may be made from and / or incorporate filaments 12 coated with a bacteriophage solution 16 as described above. Such medical products 120 and those shown in FIGS. 1-4 may include one or more filaments, each filament 12 may include multiple regions R containing the same bacteriophage 18 or multiple regions R containing different bacteriophages 18 before being formed into a finished product. The finished product may have multiple zones Z1, Z2, Z3, each zone having a different bacteriophage 18, which is facilitated by forming each zone from a different filament 12, where each filament 12 has a different bacteriophage 18 applied to it, or where the filament 12 has bacteriophage 18 in different regions R.

[0053] The method of making the product or article 10 will now be described in a flow sequence to more specifically illustrate the elements of the process. With reference to the drawings in general, and with particular reference to Figures 8 and 9, the production of a filament 12 having bacteriophage distributed on / in its surface will now be described. First, a filament of material, e.g. an artificial material, is passed through an optional cleaning / etching solution, e.g. in the form of hydrogen chloride, to prepare the surface 12a of the filament 12 prior to the subsequent steps. A next optional step includes a plasma treatment step, performed by passing the filament 12 through a plasma generator 64, to modify the surface of the filament 12, thereby making the surface 12a more hydrophilic. Droplets 54 of a first solution 16 containing a first bacteriophage are dispensed, sequentially or simultaneously, onto one or more filaments 12, such that a predetermined or preselected amount of the solution, and thus a predetermined or preselected amount of bacteriophage 18, is dispensed onto one or more regions R1-R4 on one or more filaments. This process may also involve applying a second solution 16b containing second bacteriophages 18b in a second carrier fluid 20b onto the same or different filaments 12 at positions spaced apart from one another or adjacent to one another. Such a process effectively deposits a predetermined or preselected amount of solution per unit length, and by analogy, a predetermined or preselected amount of bacteriophages per unit length. This may be on one or more filaments. Gaps G may be left between the deposited droplets 54, so that there may or may not be gaps G or separations between the deposited bacteriophages when the droplets are adsorbed or otherwise incorporated onto the surface 12a of the filament 12. The important thing to do is to apply just enough solution to each droplet 54 to allow the droplets 54 to be adsorbed to the surface 12a of the filament 12 without creating an excessive amount of waste. The droplets can be dispensed at a frequency / amount sufficient to saturate the entire length of the filament surface 12a, or at a frequency / amount insufficient to saturate the entire length, thereby forming the defined regions R and gaps G described above.Once the droplets 54 are deposited, they tend to spread as described above as they saturate the surface 12a of the filament 12. Once the droplets 54 are fully adsorbed, the filament 12 can undergo a further step of being surrounded by a cylindrical electrode 66 and applying a potential between the electrode and the filament, thereby aligning the negatively charged heads 18a of the bacteriophages 18 against the filament surface 12a and causing the legs 18b of the bacteriophages 18 to extend outwardly from the filament 12. As described above and also with reference to Figures 1-4 and 10, once the bacteriophages are aligned, the filament 12 can be on a former (not shown) for later use or can proceed immediately to use in the manufacture of the article or product 10. The method can include forming the article 10 such that the article 10 has different zones Z1, Z2, Z3, each of which can be formed from a different filament 12 having a different bacteriophage applied thereto. Such an approach would allow specific bacteriophages to be tailored to different zones Z of the final article or product, which may allow the bacteriophages to be better suited to anticipated bacterial infections in different parts of the body adjacent to the article or product 10.

[0054] It will be understood that, although not specifically claimed separately herein, the tubular electrode 66 and the method of using same to align bacteriophage 18 on the filament 12 may also form a separate invention and may be claimed in a future application. It will also be understood that, although the arrangement of Figure 8 is not claimed (in part or in whole) as an apparatus, it may also form a separate invention and may be claimed in a future application.

[0055] For the avoidance of doubt, in this specification, discrete means unitary and separate from another of the same thing.

[0056] Any system features described herein may also be provided as method features, and vice versa. As used herein, means-plus-function features may be alternatively expressed in terms of their corresponding structures.

[0057] Any feature in one embodiment may be applied to other embodiments in any suitable combination. In particular, method embodiments may be applied to system embodiments and vice versa. Furthermore, any, some, and / or all features in one embodiment may be applied to any, some, and / or all features in any other embodiment in any suitable combination.

[0058] It should also be understood that specific combinations of the various features described and defined in any embodiment may be implemented and / or provided and / or used independently.

[0059] Experimental Evidence The advantages of the present invention were demonstrated by the following experiments performed by the applicant: Filaments 12 carrying various amounts and compositions of bacteriophage 18 were exposed to various types of bacteria and the results were reviewed after overnight incubation at 37° C. The effectiveness of filaments 12 was then measured.

[0060] 1- Droplet spacing and its effect on antibacterial efficacy Groups of three 4 cm long silk filament samples each had separate droplets of 4 microliters of the same S. aureus phage solution applied to them, spread along their length with different spacing between the separate locations where the droplets were applied, and the treated filaments were compared by zone of inhibition assay.

[0061] SA-A group: All three samples were loaded with 12 4 μl droplets of SAP1 with minimal spacing between droplets.

[0062] SA-B group: All three samples were loaded with six 4 μl SAP1 droplets with equal spacing between droplets.

[0063] Group C: All three samples were loaded with four 4 μl drops of SAP1 with an equal spacing of two drops between drops.

[0064] Group D: All three samples were loaded with three 4 μl drops of SAP1 with a 1 cm spacing between drops.

[0065] All samples were placed on a lawn of one strain of Staphylococcus aureus (SA-C) and incubated overnight at 37° C. The effectiveness of filament 12 was assessed by measuring the area of ​​the lysis zone, which is the darker region in the image of FIG. 12. The surface area was measured using Image-J. [Table 1]

[0066] The results show that the lysis zone is about 10 times larger in samples with more widely spaced droplets, clearly demonstrating the advantage of pre-determining and controlling the amount of bacteriophage solution per unit length. The results also suggest possible interference between different phage deposits and the advantage of being able to very precisely control the deposition of separate droplets onto the material.

[0067] 2-Symbiotic effect of multiphages added to silk filaments A total of six treated filament groups were compared by zone of inhibition assay.

[0068] To all six groups of three silk filament samples, 4 cm in length, six separate droplets of 4 microliters of phage solution were applied, spread along the length, respectively. · For groups A, C, and E, different phage solutions (SAP1, SAP5, and SAP8), respectively, were applied in an alternating pattern of 2-2-2 drops. Only one phage solution was applied to each of groups B, D, and F (SAP1 for group B, SAP5 for group D, and SAP8 for group F).

[0069] All samples from groups A and B were placed on a lawn of a first S. aureus "SAA". All samples from groups C and D were placed on a lawn of a second S. aureus "SAC". All samples from groups E and F were placed on a lawn of a second S. aureus "SAE". The plates were incubated overnight at 37°C and then the lysis zones of each group were compared. [Table 2]

[0070] The results showed that filaments to which an alternating pattern of all three phages was applied had a larger lysis zone than filaments to which only one phage was applied. Thus, the separate deposition method can enhance the antibacterial effect while precisely controlling the amount of different phages added to the filament. This indicates the possibility that a variety of different bacteriophages18 or a cocktail of complementary bacteriophages18 can be added to different filaments12 within one and the same device, thus enabling the development of devices active against multiple types of bacteria (e.g., Staphylococcus aureus, Escherichia coli, Pseudomonas spp., etc.).

[0071] 3- Complete kill of bacteria for 7 days after 4 re-exposures to S. aureus. Three short 1 cm filaments were fabricated by depositing 4-microliter droplets of S. aureus phage solution (OSPT) and compared with short 1 cm pieces of commercially available Vicryl Plus suture (Vicryl Plus).

[0072] All samples were added to microtubes containing 200 μL of 10'5 CFU / mL S. aureus bacterial solution and placed in a 37°C incubator along with a control (Contr.) of S. aureus bacteria only for an initial period of 24 hours. After 24 hours, each sample was removed from its respective bacterial solution and placed into a new microtube containing 200 μL of S. aureus and left at 37°C for another 24 hours. Meanwhile, all extracts from the first 24 hour microtubes were diluted 9-fold, plated on agar plates (BHIA) and left at 37°C overnight. Samples were transferred to new microtubes and new bacterial solutions three times in succession, up to a total of 168 hours. [Table 3]

[0073] The results showed that there was no bacterial growth in any of the solutions to which the OSPT samples were applied, in contrast to the commercially available antimicrobial Vicryl Plus suture, where bacteria grew to the same extent as the control after 24 hours. This indicates that bacteria are completely eliminated when exposed to OSPT-treated filaments using a small, controlled, and defined volume of phage solution per unit length.

Claims

1. 1. A method for producing an article (10) comprising one or more filaments (12) of a material having a hydrophilic surface (12a), comprising: a) forming a first solution (16) containing one or more bacteriophages (18a) suspended in a carrier fluid (20); b) providing one or more first filaments (12); c) depositing the first solution (16) containing the one or more bacteriophages (18) on the one or more first filaments (12), said depositing of said first solution (16) is performed by dispensing droplets (22) thereof from a droplet dispenser (24) to deposit a predetermined amount of said first solution per unit length onto said first filament (12); forming the one or more first filaments (12) into a final product (10); A method for producing an article (10), comprising:

2. 2. The method of claim 1, further comprising the steps of: forming a second solution (16b) containing one or more second bacteriophages (18b) different from the first bacteriophage (18a) suspended in a second carrier fluid (20b); providing one or more second filaments (12b); depositing the second solution (16b) containing the one or more second bacteriophages (18b) on the one or more second filaments (12b), wherein a predetermined amount of the second solution per unit length is deposited on the second filament (12); and forming the one or more second filaments (12b) together with the first filament (12a) into a final product (10).

3. the one or more first filaments (12) each have a first region R1 distinct from a second region R2; forming a second solution (16b) containing one or more second bacteriophages (18b) different from the first bacteriophage (18a) suspended in a second carrier fluid (20b); depositing a predetermined amount per unit length of the first bacteriophage solution (16a) on the first region R1; depositing a predetermined amount per unit length of the second bacteriophage solution (16b) on the second region R2; and forming the one or more filaments (12) into a final product (10). The method of claim 1 further comprising:

4. The method of any one of claims 1 to 3, wherein the forming step to form the article (10) comprises one or more of weaving, braiding, plating, knitting, embroidering an article, or forming a nonwoven article.

5. The bacteriophage is ２ PFU / mL ~ 10 １２ 5. The method of claim 1, further comprising the step of mixing in said solution (16) a bacteriophage (18) at a concentration per unit solution of between 100 and 200 PFU / mL.

6. The bacteriophage is ５ PFU / mL ~ 10 ９ 5. The method of claim 1, further comprising mixing in said solution at a concentration of bacteriophage per unit solution of between 100 and 200 PFU / mL.

7. 7. The method of claim 1, wherein the filament (12a or 12b) has a length L, and the method comprises depositing the solution (16a or 16b) at discrete spaced locations along the length L of the filament (12a or 12b).

8. 8. The method of claim 1, comprising simultaneously depositing a plurality of droplets (30a-30d) of the solution (16a or 16b) onto the filament (12a or 12b) at distinct spaced locations along the filament (12a or 12b).

9. The method of any one of claims 1 to 8, comprising depositing the droplets (30) on the filament (12) at intervals sufficient to saturate the entire length L of the filament (12).

10. 9. The method of claim 1, further comprising depositing the droplets (30) on the filament (12) at intervals insufficient to saturate the entire length L of the filament (12), thereby forming a separate length G of the filament (12) to which no bacteriophage has been applied.

11. 11. A method according to any one of claims 1 to 10, comprising the steps of producing an article (10) having two or more zones (Z1, Z2) and forming said article (10) of filaments (12) carrying different bacteriophages, thereby forming an article (10) carrying different bacteriophages in its different zones.

12. 12. The method of any one of claims 1 to 11, further comprising forming the article (10) having an end (10a) and a non-end (10b), the article (10) having a first filament (12a) having a first bacteriophage at the end (10a) and a second filament (12b) having a second bacteriophage at the non-end.

13. 13. The method of claim 1, further comprising the step of mixing a solution containing the bacteriophage prior to deposition on the filament.

14. A method according to any one of claims 1 to 13, comprising modifying the surface of the filaments to make them more hydrophilic.

15. The method of claim 14 including the step of modifying a surface of the filament by passing the filament through a plasma discharge.

16. 16. A method according to any one of claims 1 to 15, comprising passing the filament along an electrode and applying a potential to align the bacteriophage with respect to the filament.

17. The method of claim 16, further comprising threading the filament through a tubular electrode.

18. 18. The method of any one of claims 1 to 17, wherein the first solution (16) is deposited on the filament (12) by simultaneously dispensing a plurality of droplets (22) of the first solution (16) onto the filament (12) from spaced apart droplet dispensers.

19. A method for producing a filament (12), comprising the steps of: a) forming a first solution (16) containing one or more bacteriophages (18a) suspended in a carrier fluid (20); b) providing one or more filaments (12); c) depositing the first solution (16) containing the one or more bacteriophages (18) on the one or more first filaments (12), said depositing of said first solution (16) is performed by dispensing droplets (22) thereof from a droplet dispenser (24) to deposit a predetermined amount of said first solution per unit length onto said first filament (12); A method for producing a filament (12), comprising:

20. the one or more first filaments (12) each have a first region R1 distinct from a second region R2; forming a second solution (16b) containing one or more second bacteriophages (18b) different from the first bacteriophage (18a) suspended in a second carrier fluid (20b); depositing a predetermined amount of the first bacteriophage solution (16a) per unit length on the first region R1; and depositing a predetermined amount of the second bacteriophage solution (16b) per unit length on the second region R2. The method of claim 19 further comprising:

21. The bacteriophage is ２ PFU / mL ~ 10 １２ 21. The method of claim 19 or 20, comprising mixing in said solution at a concentration of bacteriophage per unit solution of between 100 and 200 PFU / mL.

22. The bacteriophage is ５ PFU / mL ~ 10 ９ 22. The method of any one of claims 19 to 21, comprising mixing in said solution at a concentration of bacteriophage per unit solution of between 100 and 200 PFU / mL.

23. 23. The method of any one of claims 19 to 22, wherein the filament (12a or 12b) has a length L, and the method comprises depositing the solution (16a or 16b) at discrete spaced locations along the length L of the filament (12a or 12b).

24. 24. The method of any one of claims 19 to 23, comprising simultaneously depositing a plurality of droplets (30a-30d) of the solution (16a or 16b) onto the filament (12a or 12b) at distinct spaced locations along the filament (12a or 12b).

25. 25. The method of claim 19, further comprising depositing the droplets (30) on the filament (12) at intervals sufficient to saturate the entire length L of the filament (12).

26. 26. The method of claim 19, further comprising depositing the droplets (30) on the filament (12) at intervals insufficient to saturate the entire length L of the filament (12), thereby forming a separate length of the filament (12) to which no bacteriophage has been applied.

27. 27. A method according to any one of claims 19 to 26, comprising the step of mixing a solution containing the bacteriophage prior to deposition onto the filament.

28. 28. A method according to any one of claims 19 to 27, comprising modifying the surface of the filaments to make them more hydrophilic.

29. 30. The method of claim 28, including modifying a surface of the filament by passing the filament through a plasma discharge.

30. 30. A method according to any one of claims 19 to 29, comprising passing the filament along an electrode and applying a potential to align the bacteriophage with respect to the filament.

31. 31. The method of claim 30, further comprising threading the filament through a tubular electrode.

32. The method of any one of claims 1 to 31, wherein the droplets have a size greater than 0.1 μL.

33. The method of any one of claims 1 to 32, wherein the droplets have a size range between 0.2 μL and 100 μL.

34. An article (10) manufactured according to the method of any one of claims 19 to 33.

35. An article (10) comprising one or more filaments (12) of a material having a hydrophilic surface (12a) and containing one or more bacteriophages within said hydrophilic surface (12a).

36. 36. The article (10) of claim 35, comprising a plurality of different filaments (12), each having a different bacteriophage contained within its surface.

37. 37. The article of any one of claims 34 to 36, wherein the one or more filaments (12) have a plurality of first portions P1 containing the bacteriophage separated from one another by portions P2 not containing the bacteriophage.

38. 38. The article of any one of claims 34 to 37, wherein the one or more filaments (12) contain a bacteriophage along their entire length.

39. 39. The article (10) of any one of claims 34 to 38, comprising a first zone Z1 formed of a first filament (12) containing a first bacteriophage and a second zone Z2 formed of a filament containing a second bacteriophage.

40. 40. The article (10) of claim 39, wherein the first zone Z1 comprises an end (10a) of the article (10) and the second zone Z2 comprises an intermediate portion (10b) of the article (10).

41. 41. The article of any one of claims 34 to 40, wherein one or more of the one or more filaments (10) comprises a natural material.

42. 42. The article of claim 41, wherein one or more of the one or more filaments (12) comprises silk.

43. 43. The article (10) of any one of claims 34 to 42, wherein one or more of the one or more filaments (12) comprises a polymeric material.

44. 44. The article (10) of claim 43, wherein one or more of the one or more filaments (12) comprises a polymeric material having a surface (12a) that comprises a hydrophilic surface.

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