WO2015044673A1

WO2015044673A1 - Methods, materials and products for delivering biocides

Info

Publication number: WO2015044673A1
Application number: PCT/GB2014/052916
Authority: WO
Inventors: Andrea Chiu Yee CHAN; Helen Elizabeth Townley; Ian Patrick THOMPSON
Original assignee: Isis Innovation Limited
Priority date: 2013-09-30
Filing date: 2014-09-26
Publication date: 2015-04-02
Also published as: GB201317293D0

Abstract

A delivery system for a biocide, wherein: the delivery system comprises a core material(which may be porous or substantially non-porous); a biocide is located within or on the core material; and the delivery system further comprises a capping material applied to the core material so as to contain the biocide within or on the core material, and to enable exposure (e.g. release) of the biocide when the capping material is broken down or opened up. Methods of designing and/or manufacturing such a delivery system and of delivering biocides are also provided, as are examples of products embodying the delivery system.

Description

METHODS, MATERIALS AND PRODUCTS FOR DELIVERING BIOCIDES Field of the Invention

The present invention relates to methods and delivery vehicles for the controlled delivery of biocides.

Background to the Invention

A biocide is a chemical substance or microorganism that can kill, deter, render harmless or inhibit the growth of a harmful organism by chemical or biological means. Biocides are commonly used in healthcare, medicine, agriculture, forestry and industry.

The term "biocide" encompasses antimicrobials, fungicides and herbicides, for example.

Of these, antimicrobials encompass, for example, antibacterials, antifungals, antivirals, germicides, antibiotics, antiprotozoals and antiparasites.

In more detail, antimicrobials are agents that kill microorganisms (e.g. bacteria or fungi) or viruses, or inhibit their growth. Antimicrobials are often grouped according to the microorganisms they act primarily against. For example, antibacterials (e.g. antibiotics) are used against bacteria, e.g. to treat bacterial infections, or to kill unwanted microbial growth. Antifungals are used against fungi, for example to treat infections such as athlete's foot, ringworm or thrush. Antivirals are used to treat viral infections, for example.

As well as healthcare applications, antimicrobials (and biocides more generally) may be used in bio-industrial applications, for example in food packaging, or to prevent the build-up of unwanted microbial growth in recirculated metalworking fluids, water circulation systems, air conditioning systems and such like. Further applications for antimicrobials are in agrochemicals, fungicides and such like. Some conventional antimicrobial agents can be unsafe and potentially damaging for the natural environment. Furthermore, they can be unsafe for human exposure at antimicrobially-active concentrations, as they indiscriminately react with the surrounding environment and can produce toxic or carcinogenic by-products.

Moreover, some biocides are very volatile and have previously been dismissed from commercial applications because they are difficult to deliver in a controlled way. Thus, there is a need for controlled delivery of antimicrobial agents, thereby permitting improved targeting of microorganisms in an effective and efficient manner.

Summary of the Invention

According to aspects of the present invention there is provided a delivery system for a biocide as defined in the accompanying claims.

In more detail, the delivery system comprises a core material (which may be porous or substantially non-porous) within or on which a biocide is located. The delivery system further comprises a capping material applied to the core material so as to contain the biocide within or on the core material (e.g. within the pores, if the core material is porous, or adsorbed onto the surface), and to enable exposure (e.g. release) of the biocide when the capping material is broken down or opened up. Thus, exposure/release of the biocide may be controlled such that this only (or preferentially) occurs in response to breakdown of the capping material.

The system of the present invention provides an effective and efficient means for delivering a biocide (such as an antimicrobial agent) to a microorganism in a controlled manner. First, the biocide is stabilised and contained within or on the core material (e.g. within the pores, if the core material is porous, or adsorbed onto the surface). Secondly, exposure/release of the biocide is responsive to the capping material being broken down, which in turn may be responsive to the metabolic activity of a microorganism in the vicinity of the delivery system. Plant secondary metabolites represent one class of effective antimicrobial agents. Other antimicrobial agents are well known to a skilled person and equally suitable for use in the present invention.

Plant secondary metabolites are organic compounds that are not directly involved in the normal growth, development or reproduction of a plant, but which often play a role in the defence of the plant against other species. Compared to conventional antimicrobial agents they are often safer and non-damaging to the natural environment, and are safe for human exposure at antimicrobially-active concentrations. However, plant secondary metabolites are often highly hydrophobic, volatile in nature, and have low solubility in aqueous environments. Surprisingly, these otherwise potentially problematic properties are overcome or at least mitigated when said plant secondary metabolites are employed within the controlled environment of the present invention.

Thus, according to a first aspect of the invention there is provided a delivery system for a biocide, the delivery system being targeted towards a specific microorganism and comprising: a core material; a biocide suitable for combating the targeted microorganism, the biocide being located within or on the core material; and a capping material applied to the core material so as to contain the biocide within or on the core material, and to enable exposure of the biocide when the capping material is broken down or opened up; wherein the capping material comprises a degradable material selected such as to be desirable to, and consumable by, the microorganism being targeted.

The core material may be porous, having a multiplicity of pores.

According to a second aspect of the invention there is provided a delivery system for a volatile biocide, wherein: the delivery system comprises a porous core material having a multiplicity of pores; a volatile biocide is located within or on the core material; and the delivery system further comprises a capping material applied to the core material so as to contain the biocide within or on the core material, and to enable exposure of the biocide when the capping material is broken down or opened up.

The use of a porous core material is particularly effective at stabilising a volatile biocide.

In certain presently-preferred embodiments the core material is mesoporous (i.e. with a pore diameter in the range of 2 nm to 50 nm). However, in other embodiments other pore diameters are possible. For example, the core material may be microporous (with a pore diameter less than 2 nm) or may be macroporous (with a pore diameter greater than 50 nm).

According to a third aspect of the invention there is provided a delivery system for a biocide, wherein: the delivery system comprises a substantially non-porous core material; a biocide is adsorbed onto the surface of the core material; and the delivery system further comprises a capping material applied to the core material so as to contain the biocide on the core material, and to enable exposure of the biocide when the capping material is broken down or opened up. In certain embodiments according to this aspect of the invention the biocide may be volatile prior to being adsorbed onto the surface of the core material, although in alternative embodiments it may be non-volatile prior to adsorption.

In embodiments according to the first, second or third aspects of the invention, the core material may be in the form of a multiplicity of discrete particles - for example, a multiplicity of nanoparticles - such that the delivery system is particulate in form, with each particle being separately capped.

In one embodiment, the nanoparticles are of the order of 100 nm in diameter. By stabilising the biocide in or on nano-sized capsules, this provides an efficient and effective delivery system for controlling unwanted microbial growth.

In one embodiment the nanoparticles are mesoporous nanoparticles, of the order of 100 nm in diameter, and containing pores of the order of 2 nm in diameter. The channels that form the nanosized pores within the mesoporous nanoparticles act as physical, protective barriers that can surround and contain volatile antimicrobial molecules (such as natural plant compounds) within the pores; hence, the mesoporous nanoparticles are capable of stabilising volatile antimicrobial agents, minimising their loss through vaporisation, and enhancing the effectiveness of each antimicrobial treatment by retaining more of the loaded agent in the system. Also, a high surface area to volume ratio, along with the mesoporous nanoparticles' accessible reaction sites on pore surfaces, also help to maximise the bio-availability of the loaded antimicrobial agent during its exposure to microorganisms.

In one embodiment the mesoporous nanoparticles are mesoporous silica nanoparticles - for example, silica nanoparticles which are inorganic, biocompatible and biodegradable. Other suitable mesoporous materials include, for example, mesoporous carbon.

In another embodiment, the core material may comprise a substrate or be part of an article of manufacture. In certain embodiments according to the first, second or third aspects of the invention, the core material is able to be attracted by a magnet. For example, the core material may comprise iron. This advantageously enables the core material (particularly if it is particulate in form) to be recovered after use, via magnetic attraction, and then potentially recharged with biocide, recapped and reused.

In embodiments according to the first, second or third aspects of the invention, the capping material may comprise a degradable material (e.g. a biodegradable material), in particular one selected such as to be desirable to, and consumable by, a microorganism (for example, a targeted microorganism). As a consequence, the microorganism is effectively lured into consuming the degradable material, causing the controlled release of the biocide in the vicinity of the targeted microorganism. In this targeted manner, the delivery system improves the "kill efficiency" per unit of biocide released. In certain embodiments, the degradable material is metabolised by the microorganism and/or broken down via extracellular secretions (e.g. enzymes such as proteases) from the microorganism.

In certain embodiments the degradable material may comprise a sugar, such as lactose, xylose, sucrose or starch - individual examples of these being attractive to certain types of bacteria.

In certain embodiments, the degradable material is selected to comprise or consist of a component that will be preferentially consumed (e.g. metabolised) by a particular class or sub-class of microorganism, thereby providing a 'targeting' element. Thus, the degradable material is selected such as to be desirable to, and consumable by, the microorganism being targeted; and such as to be relatively undesirable to, and relatively unconsumed by, microorganisms not being targeted. Alternatively and/or in addition the same 'targeting' effect may be achieved by use of one or more specific attractants in, or in close proximity to, the degradable material.

Alternatively the degradable material may comprise a material that is soluble in the medium into which it is introduced in use (i.e. in the vicinity of the targeted microorganism). This has particular application in healthcare products such as handwashes and disinfectants, whereby water is used to break down the degradable material to release the biocide (e.g. an antimicrobial agent) at the point of use. In this manner the antimicrobial agent is preserved within the mesoporous material until it is required for use, thereby maximising its efficacy.

In other alternative embodiments, the degradable material is able to be broken down or opened up by the effect of one or more other physical, chemical or biological processes, such as (but not limited to): ultrasound, irradiation, light, UV light, ionic change, pH level, temperature, or osmotic change. With at least some of these processes, an external trigger (e.g. activating an ultrasound emitter or a source of irradiation, etc.) may advantageously be used to initiate the breakdown of the degradable material and cause the exposure of the biocide, as and when required. The said effect may be automatically triggered, without human intervention, by control means in response to receiving an activation signal (e.g. an electrical signal). The activation signal may be provided by a sensor, which may be, for example, a detector of a particular undesired microorganism. The delivery system may also contain a dye that is arranged to be released from the core material when the capping material is broken down or opened up, thereby enabling an observer to detect where and when the biocide is released, and in what quantity. In certain embodiments the biocide comprises an antimicrobial agent. Advantageously, the antimicrobial agent may comprise a plant secondary metabolite, so as to be safe and non-damaging to the natural environment, and safe for human exposure at antimicrobially-active concentrations. The plant secondary metabolite may comprise, for example, allyl isothiocyanate or cinnamaldehyde. Many other plant secondary metabolites are also promising for use as antimicrobial agents, or may be discovered in the future - to which the present work is applicable.

Such plant secondary metabolites are relatively volatile. It will therefore be appreciated that the present work enables the controlled introduction of biocides that are relatively volatile (thereby increasing the range of chemicals that can be exploited to kill microorganisms) and which have previously been dismissed because they are so difficult to deliver in a controlled way. Hence, a small aliquot of biocide can be released in a very large volume of solution, but targeted exactly where it is required - in the direct proximity of the cells which have removed the capping material. Thus less biocide is required since it is strategically delivered exactly where it is required.

The antimicrobial agent may be an antibacterial agent, or an antifungal agent, or an antiviral agent.

According to a fourth aspect of the invention there is provided a method comprising: identifying a microorganism to be targeted; identifying a biocide suitable for combating the microorganism to be targeted; identifying a degradable material that is desirable to, and consumable by, the microorganism to be targeted; and designing a delivery system for the said biocide, wherein: the delivery system comprises a core material; the said biocide is located within or on the core material; and the delivery system further comprises a capping material comprising the said degradable material, applied to the core material so as to contain the biocide within or on the core material, and to enable exposure of the biocide when the capping material is broken down or opened up. The method may further comprise manufacturing the delivery system.

According to a fifth aspect of the invention there is provided a method of manufacturing a delivery system for a volatile biocide, the method comprising: providing a porous core material having a multiplicity of pores; introducing a volatile biocide into or onto the core material; and applying a capping material to the core material so as to contain the biocide within or on the core material, such that the biocide is able to be exposed when the capping material is broken down or opened up. According to a sixth aspect of the invention there is provided a method of manufacturing a delivery system for a biocide, the method comprising: providing a substantially non-porous core material; introducing a biocide onto the core material such that the biocide is adsorbed onto the surface of the core material; and applying a capping material to the core material so as to contain the biocide on the core material, such that the biocide is able to be exposed when the capping material is broken down or opened up. In certain embodiments according to this aspect of the invention the biocide may be volatile prior to being adsorbed onto the surface of the core material, although in alternative embodiments it may be nonvolatile prior to adsorption.

Optional features of the fourth, fifth and sixth aspects are as described above in relation to the first, second and third aspects of the invention. According to a seventh aspect of the invention there is provided a method of delivering a biocide, the method comprising: providing a delivery system in accordance with the first, second or third aspects of the invention; introducing the delivery system to a microorganism or to an environment to be protected from said microorganism; and allowing the capping material to break down or be opened up, thereby releasing the biocide from the core material.

As mentioned above, the degradable material may be desirable to and consumable by the microorganism, such that the microorganism consumes at least some of the capping material and thereby releases the biocide in a targeted manner.

Alternatively the capping material may be at least partially soluble in a medium in the vicinity of the microorganism, and the method may further comprise introducing the delivery system to the medium such that the capping material breaks down and thereby releases the biocide. Such a technique may be employed when dispensing an antibacterial handwash or disinfectant embodying the invention, for example. In further alternatives, the method may further comprise breaking down or opening up the capping material by the effect of one or more other physical, chemical or biological process, such as (but not limited to): ultrasound, irradiation, light, UV light, ionic change, pH level, temperature, or osmotic change. With at least some of these processes, an external trigger (e.g. activating an ultrasound emitter or a source of irradiation, etc.) may be used to initiate the breakdown of the degradable material and cause the exposure of the biocide, as and when required. The said effect may be automatically triggered, without human intervention, by control means in response to receiving an activation signal (e.g. an electrical signal). The activation signal may be provided by a sensor, which may be, for example, a detector of a particular undesired microorganism.

After use, in the case of variants in which the core material is able to be attracted by a magnet, the method may further comprise attracting the core material using a magnet, thereby enabling the core material to be potentially recharged with biocide, recapped and reused.

Further aspects of the invention provide healthcare products (e.g. an antibacterial handwash or disinfectant, or an antifungal product, or an antiviral product), bio- industrial products (e.g. for preventing build-up of unwanted microbial growth in metalworking fluids, water circulation systems, air conditioning systems and such like, or for use in agriculture), and biocide delivery products more generally. Brief Description of the Drawings

Embodiments of the invention will now be described, by way of example only, and with reference to the drawings in which:

Figure 1 schematically illustrates a hexagonally-ordered mesoporous silica nanoparticle (the pores extending into the page), covered with a biodegradable capping material;

Figure 2 schematically illustrates a cross-sectional view through part of the mesoporous silica nanoparticle of Figure 1 (the pores extending in the plane of the page), showing the capping of the pores by the biodegradable material;

Figure 3 is a typical TEM (transmission electron microscopy) image of some mesoporous silica nanoparticles (MSNs);

Figure 4 is a TEM image displaying two adjacent MSNs;

Figure 5 is a TEM image of an MSN having pores aligned in a hexagonal pattern; Figure 6 is a plot showing the size distribution of MSNs obtained from measurements of TEM images;

Figure 7 is a plot showing the release profile of calcein from 200 μΙ aliquots of 20 mg calcein-loaded MSNs in 1 .5 ml of phosphate buffered saline (PBS);

Figure 8 is a plot showing the release profile of allyl isothiocyanate (AIT) from 20 mg AIT-loaded MSNs in 10 ml of PBS; and

Figure 9 is a plot showing the release profile of cinnamaldehyde (CNAD) from 20 mg CNAD-loaded MSNs in 10 ml of PBS.

Figures 1 and 2 are not to scale. Detailed Description of Preferred Embodiments

The present embodiments represent the best ways known to the applicants of putting the invention into practice. However, they are not the only ways in which this can be achieved.

Overview

A novel delivery system for a biocide is provided. The delivery system is particularly applicable, but by no means limited, to the delivery of a volatile biocide, such as a plant secondary metabolite.

The delivery system comprises a core material (which may be porous or substantially non-porous; and may be particulate, or not). A biocide (e.g. an antimicrobial agent) is located within or on the core material. Thus, it will be appreciated that the core material is distinct from the biocide. The core material provides a means for carrying the biocide, without the core material modifying the biocide.

The delivery system further comprises a capping material applied to the core material so as to contain the biocide within or on the core material, and to enable exposure (e.g. release) of the biocide when the capping material is broken down or opened up.

The capping material is selected such as to be broken down by (or in the presence of) the microorganism being targeted, causing release of the biocide directly in the vicinity of the targeted microorganism. The capping material may be biodegradable (in particular, intended to be consumed by the microorganism being targeted) or environmentally degradable (e.g. soluble in the medium into which it is introduced in use). Through the use of such (bio)degradable material, this provides an effective and efficient way of targeting the intended microorganism with the biocide and, in turn, potentially reduces the dosage levels required.

This delivery system is particularly suitable for the release of a plant secondary metabolite as an antimicrobial agent, since the capping of the antimicrobial agent by the degradable material contains the antimicrobial agent until it is required to be released, thereby overcoming (or at least mitigating) issues associated with the hydrophobicity, volatile nature and low solubility of the antimicrobial agent. Example embodiments

According to a presently-preferred embodiment the delivery system is particulate in form, with the core material comprising a multiplicity of hexagonally-ordered mesoporous silica nanoparticles (MSNs). Silica nanoparticles are inorganic, biocompatible and biodegradable.

Figure 1 schematically illustrates a particle 10 comprising a MSN 12 coated with a biodegradable capping material 16. The MSN 12 contains a multiplicity of pores 14 arranged in a substantially hexagonal configuration (as illustrated, the pores extend into the plane of the page). The pores 14 are loaded with an antimicrobial agent and are capped by the capping material 16, thereby sealing the antimicrobial agent inside the pores 14. Figure 2 schematically illustrates a cross- section through part of the particle 10, showing that pores 14 may extend through the entire thickness of the MSN 12 and may be capped at both ends by the capping material 16. It should be noted that the shapes shown in Figures 1 and 2 are merely schematic, and that, in practice, the particles 10 may have a shape similar to the MSNs 12 shown in Figures 3 to 5 (as discussed in more detail below).

As used in the present work, the diameter of each MSN 12 is of the order of 100 nm, and the diameter of each of the constituent pores 14 is of the order of 2 nm. The synthesis of the MSNs is described below, together with a characterisation process that has been carried out.

In the present work, the antimicrobial agent is loaded into the pores 14 by diffusion, prior to the biodegradable capping material 16 being applied. Preferably the antimicrobial agent has an attraction to the material from which the mesoporous nanoparticles 12 are made (e.g silica), in order to facilitate the loading of the antimicrobial agent into the pores 14. The particle size, porosity, and surface properties of the MSNs 12 can be predictably controlled and tailored to match the physicochemical properties of the antimicrobial agent as well as the overall needs of specific applications. Functional groups, targeting ligands, and stimuli-sensitive molecules can be conjugated to the surfaces of the pores 14 and the particles themselves, in order to optimise loading of the antimicrobial agent, control the release profile, improve dispersity, and direct the carriers to targeted sites. In an embodiment targeted at bacteria, the biodegradable pore capping material 16 comprises a sugar such as lactose, selected so as to be desirable for the bacteria to consume, and the antimicrobial agent may be a plant secondary metabolite. Lactose molecules can be conjugated onto the nanoparticle surface to serve as pore caps for controlled release of the antimicrobial agent. The lactose is degraded by the bacteria, resulting in release of the antimicrobial agent immediately adjacent to the cells the agent is targeted to kill. This targeted nature of the biocide release improves the "kill efficiency" per millilitre of biocide released.

It will therefore be appreciated that the capping material 16 enables the pores 14 to carry hydrophobic, volatile, plant-derived antimicrobial agents with increased efficiency over free agents, and enables release of the antimicrobial agent controlled by the targeted bacteria themselves, as the bacteria consume the capping material 16. Sugar, as a capping material, has a further advantage of being recyclable.

The particles 10 can be regarded as analogous to a "Trojan horse", whereby the capping material 16 is desirable to the targeted microorganism (e.g. bacteria), so the microorganism consumes it. However, on doing so, this causes the antimicrobial agent contained within the pores 14 to be released, killing the microorganism or inhibiting its growth. For any given application, the capping material 16 is selected or tailored so as to be desirable to the microorganism being targeted, or so as to be broken down in its presence. With particular reference to Figure 2, the channels that form the nanosized pores 14 within the MSNs 12 act as physical, protective barriers that surround and contain volatile antimicrobial molecules (such as natural plant compounds) within the pores 14; hence, MSNs 12 are capable of stabilising volatile antimicrobial agents, minimising their loss through vaporisation, and enhancing the effectiveness of each antimicrobial treatment by retaining more of the loaded agent in the system. Also, the high surface area to volume ratio, along with the MSNs' accessible reaction sites on pore surfaces, also help to maximise the bioavailability of the loaded antimicrobial compound during its exposure to microorganisms.

Selection of the capping material

To illustrate in more detail the way in which embodiments can selectively target certain microorganisms in preference to others, take for example lactose-capped particles. Species of bacteria having the lac operon are able to metabolise lactose, whereas species lacking the lac operon will not break down lactose.

Pseudomonas, for example, do not utilise lactose. On the other hand, due to presence of the lac operon, E.coli is able to metabolise lactose. So a mixture which contains a mixture of Pseudomonas (which are mostly non-pathogenic) and E.coli (which is pathogenic) could be differentiated in terms of kill, since the latter does assimilate lactose. In other words, lactose-capped particles are suitable for combating E.coli in preference to Pseudomonas. As another example, Prokaryotes that possess an isomerase pathway and two oxidative pathways (called Weimberg and Dahms pathways) are able to metabolise D-xylose, whereas microbes lacking these pathways are unable to break down xylose. Thus, xylose-capped particles are suitable for combating xylose-consuming Prokaryotes in preference to other microbes which do not.

As a further example, the assimilation of carbon sources can be used to differentiate different bacterial species, for example:

Corynebacterium diptheria gravis - uses starch but not sucrose Corynebacterium diphtheria mitis - does not use starch nor sucrose

Corynebacterium xerosis - assimilates both sucrose and starch

Some strains of Staphylococcus are pathogenic and others represent part of the microflora of the skin. These can be differentiated on the basis of assimilation of mannitol and/or trehalose.

Thus, it is possible to engineer a particle which just combats key 'bad' microorganisms, and allows the 'good' microorganisms to survive - similar to probiotics.

In effect, therefore, the present work provides a method comprising: identifying a microorganism to be targeted; identifying a biocide suitable for combating the microorganism to be targeted; identifying a degradable material that is desirable to, and consumable by, the microorganism to be targeted; and designing a delivery system for the said biocide, wherein: the delivery system comprises a core material, the said biocide is located within or on the core material, and the delivery system further comprises a capping material comprising the said degradable material, applied to the core material so as to contain the biocide within or on the core material, and to enable exposure of the biocide when the capping material is broken down or opened up. The designed delivery system can then be manufactured and subsequently used.

Synthesis of MSNs with hexagonally-ordered mesopores

In the present work, MSNs were synthesised via hot hydrolysis in an aqueous, base-catalysed sol-gel system as described in Horn et al. (C. Horn et al. , "Mesoporous silica nanoparticles facilitate delivery of siRNA to shutdown signaling pathways in mammalian cells", Small, 2010. 6: pp 1 185-1 190, 2010), with some modifications. In a typical synthesis, 200 mg CTAB (surfactant) (Aldrich, 99%, UK) was dissolved in 96 ml of deionised water and 700 μΙ of 2M NaOH in a 250-ml round-bottom flask at 80^°C (pH 12.4) with stirring at 500 rpm. Once the temperature stabilised, 1 ml of TEOS (silica precursor) (Aldrich, UK) was added into the system. After stirring for 15 minutes, 254 μΙ of 3- (trihydroxysilyl)propylmethylphosphonate (THPMP) was added to the suspension in order to modify the silica surface with phosphonate groups to reduce aggregation. After stirring for another 2 hours at 80^°C, the suspension was centrifuged at 9000 rpm for 5 minutes. The resulting nanoparticles that were collected were washed twice with methanol (Aldrich, UK). The surfactant CTAB was subsequently removed by refluxing in 40 ml of methanol plus 2 ml of 37% hydrochloric acid (HCI) (Aldrich, UK) overnight at 80^°C using a water-cooled coil condenser. Characterisation of the mesoporous silica nanoparticles

The MSNs were imaged with bright field transmission electron microscopy (TEM) in different orientations to determine the shape and dimension of the particles as well as the pores. The mesopores showed high rotational symmetry about the centre axis in a hexagonal arrangement (a central pore surrounded by six equidistant pores) when imaged through the axis of the central pore, which will be denoted as "spot contrast" view. In the "line contrast" view, MSNs displayed line array of pores running through the entire volume of the nanoparticle when the electron beam was perpendicular to the centre axis. From such images, a synthesised MSN may be described as a polyhedron with constituent pore channels arranged in cubic order. It is common for MSNs with pores orientated with the electron beam (spot contrast view) to show a more circular projection as compared to those with pores orientated transversely across the beam (line contrast view), which appear more elongated. Figure 3 shows a typical TEM image of some MSNs. Arrow A indicates an MSN in spot contrast view, imaged with pores orientated along the electron beam; spherical in shape. Arrow B indicates an MSN in spot contrast view, imaged with pores orientated transversely across the electron beam, in an elongated "kidney" shape.

Figure 4 shows a TEM image displaying two adjacent MSNs, one in spot contrast view (arrow A) with pores arranged hexagonally, and the other (arrow B) with a line array of pores running through the entire volume of the nanoparticle. Figure 5 shows a TEM image of an MSN showing pores aligned in a hexagonal pattern, the arrow pointing to the centre axis of the pattern. The MSN particle diameters were measured using histograms generated by building a contrast profile along selected regions on TEM images. The distribution of nanoparticle size is displayed in Figure 6. 140 particle diameters were measured. The mean particle size was 103.64 ± 18.78 nm in diameter. The average aspect ratio was determined to be 1 .12. The size distribution of the MSNs follows a unimodal Gaussian curve with standard deviation of 18.78, which means the synthesis method was robust and reproducible, yielding particles of similar size with expected deviation.

The mean pore size in the MSNs was also determined using TEM. MSN pores were measured from TEM images using the same protocol as that for determining particle size. The mean pore size was 2.03 ± 0.19 nm in diameter (170 pores from 15 different nanoparticles being measured). The mean channel width was 2.02 ± 0.17 nm in diameter (70 channels from 15 different nanoparticles being measured). These pore size and channel width measurements are highly consistently with each other, and the low standard deviations for both are indicative of the uniformity and regularity of the mesopores.

Finally, using a Gemini VI Surface Area Analyzer, the surface area of the MSNs was found to be 1022.78 ± 3.22 m²/g based on the multilayer BET theory, and 1671 .28 ± 66.67 m²/g based on the monolayer Langmuir theory (these theories both being well known to those skilled in the art of the characterisation of porous materials).

Loading and release demonstrations

As proof of concept to show that compounds can be loaded into the pores 14 of the MSNs 12, and can subsequently provide sustained release over time, the following demonstrations have been carried out, using (separately) calcein, allyl isothiocyanate (AIT), and cinnamaldehyde (CNAD) as "guest compounds" or "guest molecules". AIT and CNAD are plant secondary metabolites that are promising candidates as antimicrobial agents.

In each of these three cases, the guest compound was loaded into the pores 14 via diffusion. As those skilled in the art will appreciate diffusion is a transport mechanism in which flux naturally goes from a region of higher concentration to a region of lower concentration until diffusive equilibrium is reached, at which the concentrations of the diffusing substance in the two regions become equal. Calcein

Calcein (C30H26N2O13, molecular weight of 622.55 g/mol) is a fluorescent dye with excitation/emission maxima of 495/515 nm, respectively. Calcein was selected as a model compound to be loaded into the synthesised MSNs because its release from the MSNs can easily be monitored by fluorescence readings over time. Calcein was chosen especially for its self-quenching property even at concentrations below 100 mM, which is important because the dye will only fluoresce when released out from the nanoparticles and not when it is dwelling inside the mesopores. This ensures an accurate release profile of the dye, in which the fluorescence measurements correlate only to the amount of calcein released.

Calcein salt (18 mg) was first dissolved in 2 ml of dimethyl sulfoxide (DMSO), and then loaded into 20 mg MSNs in 7 ml of phosphate buffered saline (PBS) via diffusion in a 15 ml glass vial. Contents were sonicated with a probe for 3 minutes at 5 second intervals, then left to stir at 250 rpm for 24 hours in the dark. After loading, the MSNs were transferred into six 1 .5-ml microfuge tubes and centrifuged at 120,000 rpm for 3 minutes. The MSNs were washed twice with 1 .3 ml of 50:50 (v:v) methanol:de-ionised water (DW) and once with 1 .3 ml of PBS to remove any excess calcein on surface of the MSNs. The washed MSNs were then resuspended in 1 .5 ml of fresh PBS and 200 μΙ aliquots of this suspension were dispensed into a 96-well black microplate (Nunc, UK). To demonstrate the subsequent release of the calcein from the pores 14, the fluorescence signal of the calcein was measured for 12 hours at 30 minute intervals, at excitation and emission wavelengths of 485/20 nm and 528/20 nm respectively.

The recorded fluorescence values are presented in Figure 7. The profile shows a gradual increase of calcein in solution over time with no spike in release at any point. The rate of release remained constant from 0 to 5.5 hours, then decelerated at 5.5 hours and remained relatively stable until 12 hours. Sustained release of calcein from the synthesised MSNs was therefore achieved, and we therefore progressed onto loading the MSNs with plant compounds AIT and CNAD.

AIT and CNAD

250 μΙ of AIT was dissolved in 1 .75 ml of DMSO, and then loaded into 20 mg of MSNs in 7 ml of PBS using the same procedure as for calcein above. In the case of CNAD, 500 μΙ of CNAD was dissolved in 1 .5 ml of DMSO, and a similar procedure as for calcein was again followed.

The loaded MSNs were washed with 50:50 (v:v) methanol: DW as described previously. The washed MSNs were resuspended in 10 ml of PBS in 15-ml centrifuge tubes. The resulting AIT-loaded and CNAD-loaded MSN suspensions were incubated at 30^°C at 150 rpm agitation.

Release was monitored every 30 minutes for 4 hours, and then again at 24 hours, via liquid-liquid extraction and subsequent GC-FID (gas chromatography - flame ionization detector) analysis. Five separate release experiments were performed for each plant compound, and three samples per timepoint were prepared in each experiment.

Liquid-liquid extraction is a method of extracting a substance from one liquid phase to another, based on relative solubilities in two different immiscible liquids such as an aqueous solution and an organic solvent. In our demonstrations, ethyl acetate (EtOAc) was used as the organic solvent to extract AIT and CNAD from the PBS solution because (i) both hydrophobic plant compounds are more soluble in EtOAC than in aqueous PBS solution, and (ii) EtOAc is a compatible solvent to be injected into the gas chromatography directly after sampling for identification and quantification. At each sampling time point, 550 μΙ of MSN suspension was transferred into a 1 .5- ml microfuge tube and centrifuged at 13,000 rpm for 2 minutes. The PBS supernatant (500 μΙ) was then transferred to a new microfuge tube, in which 500 μΙ (equal volume) of EtOAc was added. This 1 : 1 (v:v) EtOAc:PBS solution was shaken at 220 rpm for 20 minutes to extract the plant compounds out from aqueous solution into the organic phase. Samples were then centrifuged at 130,000 rpm for 2 minutes to separate the two immiscible liquids, and 220 μΙ of the top EtOAc layer was transferred into a GC vial. The sample was then injected into the GC-FID instrument to be analysed. A GC-2010 (Shimadzu) GC-FID instrument with Perkin Elmer Clarus GC ovens was used to quantify the release of AIT and CNAD from 20 mg samples of loaded MSNs.

The areas under the curve of the relevant peaks of the AIT GC-FID chromatographs were translated into concentrations of AIT detected in solution (employing an experimentally-derived GC-FID calibration curve of AIT). The resulting release profile of AIT is presented in Figure 8.

Likewise, the areas under the curve of the relevant peaks of the CNAD GC-FID chromatographs were translated into concentrations of CNAD detected in solution (employing an experimentally-derived GC-FID calibration curve of CNAD). The resulting release profile of CNAD is presented in Figure 9.

The release of both AIT and CNAD from the MSNs seemed to be rapid at the start, reaching peak concentrations at 1 hour. The high initial rates of release (AIT: 430.43 mg/L/h, CNAD: 40.02 mg/L/h) may be due to the extreme concentration gradient established from within the pores and outside the pores in the external solution. Slight and gradual decrease in concentration was detected from 1 to 24 hours for both compounds. This decrease was most likely attributed to the highly volatile nature of both plant compounds, since both are easily lost from the system via spontaneous vapourisation. Release of AIT or CNAD may have still persisted from 1 hour onwards, but the compounds' rate of escape into the atmosphere may have been greater than the rate of their release from the MSNs. It is believed that, without sustained release of the plant compounds from the MSNs compensating for the loss through vapourisation, the concentrations of AIT and CNAD at 24 hours may have been much lower than those detected in these experiments Much higher concentrations of AIT were detected in solution than CNAD (approximately ten-fold higher) as a result of diffusive release from the same amount of MSNs (20 mg). This may be due to a combination of several factors: (1 ) AIT, a C3-N-C linear molecule with molar mass of 99.15 g/mol, is a smaller molecule than CNAD, an C9 molecule with an aromatic ring structure with molar mass of 132.16 g/mol, and therefore more AIT molecules can be housed in the mesopores of MSNs as compared to CNAD molecules; (2) again, because AIT is a smaller molecule than CNAD, AIT may have been released more readily from the pore openings due to less physical self-obstruction than CNAD had; (3) because AIT is more volatile and hydrophobic than CNAD, AIT may have preferentially diffused into the pores of the MSNs more effectively than CNAD did in order to partition away from the aqueous PBS solution during loading; and (4) because AIT is more "sticky" and hydrophobic than CNAD, AIT may have clung onto the surface of the MSNs more strongly than CNAD did (even after three washes with 50:50 (v:v) methanol: DW), and therefore the amount of AIT or CNAD detected as release from the pores actually includes release from the surfaces of the MSNs as well. The release profiles of AIT and CNAD were quite distinct from that of calcein, which is not surprising since the physicochemical properties of the plant compounds and the fluorescent dye are quite different. In conclusion, therefore, this work demonstrates that the sol-gel method of MSN synthesis yielded uniform and regular nanoparticles of ~100 nm in diameter with pores of ~2 nm in diameter. Furthermore, the results of loading and release experiments using calcein, AIT, and CNAD as guest compounds showed that MSNs facilitated sustained release and maintenance of these compounds in solution.

Applications

The particles 10 described above can be used to kill unwanted microbial growth, in both planktonic and biofilm forms.

In the healthcare sector, applications include antibacterial handwashes and disinfectants, whereby water is used to break down the capping material 16 to release the antimicrobial agent at the point of use. For such healthcare applications, a sugar capping material 16 may be used.

Many other healthcare applications are also possible, as those skilled in the art will appreciate, e.g. for other antibacterial, antifungal or antiviral purposes.

Other applications, in the bio-industrial sector, include products for preventing build-up of unwanted microbial growth in recirculated metalworking fluids, water circulation systems, air conditioning systems (e.g. to prevent Legionella), and such like. Agricultural applications are also envisaged. In the case of industrial solutions such as metalworking fluids, existing biocide rules are restrictive, and there is a desire to replace slow-release formaldehyde. The present work provides a way of achieving this.

Many further applications for this technology will be apparent to those skilled in the art in light of the present disclosure.

Summary

No previous reports are known of plant secondary metabolites being encapsulated in mesoporous silica nanoparticles for the purpose of killing microbes. The present combination of plant compounds and MSNs is a new antimicrobial strategy that is (1 ) safer and non-damaging for the natural environment and (2) safe for human exposure at antimicrobially-active concentrations, unlike current antimicrobial approaches that indiscriminately react with surrounding environment and produce toxic/carcinogenic by-products. Furthermore, (3) the controlled release of the biocide by the target bacteria themselves is particularly beneficial.

An advantageous feature of MSNs is that particle size, porosity, and surface properties can be predictably controlled and tailored to match the physicochemical properties of guest compounds as well as the overall needs of specific applications. Functional groups, targeting ligands, and stimuli-sensitive molecules can be conjugated to surfaces of pores and particles themselves in order to optimise loading of guest compounds, control the release profile, improve dispersity, and direct the carriers to targeted sites. Thus, employment of MSNs to deliver antimicrobial compounds can decrease the amount of agent used in each dose because less of the compound will be lost through non-specific diffusion to areas with no cells. As described above, pores of MSNs can be capped with biodegradable molecules to control the release of loaded compounds in response to surrounding microenvironment. Of particular interest presently is to cap pores with sugar moieties to seal antimicrobial plant compounds within the nano-spheres. The loaded MSNs in this state are antimicrobially inactive, avoiding issues of over- exposure which would typically lead to resistance development. Upon exposure to specific microorganisms, the carbohydrate caps are metabolically degraded by the target microbes themselves in a "Trojan horse" self-killing approach. Such nanoparticles are thus a good means of delivering toxic doses of plant-derived antimicrobial agents to undesired microbes.

Other possible modifications and alternatives

Detailed embodiments have been described above. As those skilled in the art will appreciate, a number of additional modifications and alternatives can be made to the above embodiments whilst still benefiting from the inventions embodied therein.

For example, the core material may be able to be attracted by a magnet (e.g. by virtue of the core material containing iron). This advantageously enables the core material (particularly if it is particulate in form) to be recovered after use, via magnetic attraction, and then potentially recharged with biocide, recapped and reused. In the above embodiments, mesoporous silica nanoparticles (MSNs) were used as the core material. However, other suitable porous particles may be used instead as the core material - for example, made of mesoporous carbon, or other materials. The porous material may be selected so as to be attractive to the antimicrobial agent, in order to facilitate the loading of the antimicrobial agent into the pores.

Instead of using particles engineered around porous core material, particles having a substantially non-porous core material may be used instead. In such cases, the biocide may be adsorbed onto the surface of the core material, prior to the application of the capping material.

Moreover, instead of using particles, other core materials (which may be porous or substantially non-porous) may be used to contain the antimicrobial agent, or onto which the antimicrobial agent is applied - again with a suitably-selected capping material being applied to seal the antimicrobial agent on or within the core material, and to enable release of the antimicrobial agent when the capping material is consumed by the microorganism being targeted, or is broken down in its presence. For example, the core material may be provided as a substrate, or may form part of an article of manufacture.

In the case of a substantially non-porous core material, the biocide may be adsorbed onto the surface of the core material, prior to the application of the capping material. The biocide may be volatile (although it need not be) prior to being adsorbed onto the surface of the core material.

Within the capped core material, a dye may be contained together with the biocide. The dye may be released simultaneously with the biocide, thereby enabling one to detect where and when the biocide is released, and in what quantity.

In the above embodiments, the effect of a microorganism consuming the capping material is employed to expose the biocide contained beneath the capping material. However, in other alternatives, the capping material may be broken down or opened up by the effect of one or more other physical, chemical or biological processes, such as (but not limited to): ultrasound, irradiation, light, UV light, ionic change, pH level, temperature, or osmotic change. With at least some of these processes, an external trigger (e.g. activating an ultrasound emitter or a source of irradiation, etc.) may be used to initiate the breakdown of the degradable material and cause the exposure of the biocide, as and when required. The said effect may be automatically triggered, without human intervention, by control means (e.g. a microprocessor) in response to receiving an activation signal (e.g. an electrical signal), which may be provided by a sensor (e.g. a detector of a particular undesired microorganism).

With regard to applications, the capping technology disclosed herein can be applied to core material having any of a variety of antimicrobial agents or biocides more generally. The technology can be used in a number of different areas, e.g. recirculated metalworking fluids, water circulation systems, air conditioning systems, or healthcare.

Various other modifications and alternatives will be apparent to those skilled in the art and will not be described in further detail here.

Claims

1 . A delivery system for a biocide, the delivery system being targeted towards a specific microorganism and comprising:

a core material;

a biocide suitable for combating the targeted microorganism, the biocide being located within or on the core material; and

a capping material applied to the core material so as to contain the biocide within or on the core material, and to enable exposure of the biocide when the capping material is broken down or opened up;

wherein the capping material comprises a degradable material selected such as to be desirable to, and consumable by, the microorganism being targeted.

2. A delivery system as claimed in claim 1 , wherein the core material is porous, having a multiplicity of pores.

3. A delivery system for a volatile biocide, wherein:

the delivery system comprises a porous core material having a multiplicity of pores;

a volatile biocide is located within or on the core material; and

the delivery system further comprises a capping material applied to the core material so as to contain the biocide within or on the core material, and to enable exposure of the biocide when the capping material is broken down or opened up.

4. A delivery system as claimed in claim 2 or claim 3, wherein the core material is mesoporous.

5. A delivery system as claimed in claim 4, wherein the core material contains pores having a diameter in the range of 2 nm to 50 nm.

6. A delivery system as claimed in claim 5, wherein the pores are of the order of 2 nm in diameter.

7. A delivery system for a biocide, wherein:

the delivery system comprises a substantially non-porous core material; a biocide is adsorbed onto the surface of the core material; and

the delivery system further comprises a capping material applied to the core material so as to contain the biocide on the core material, and to enable exposure of the biocide when the capping material is broken down or opened up.

8. A delivery system as claimed in claim 7, wherein the biocide is volatile prior to being adsorbed onto the surface of the core material.

9. A delivery system as claimed in any preceding claim, wherein the core material is in the form of a multiplicity of discrete particles, such that the delivery system is particulate in form.

10. A delivery system as claimed in claim 9, wherein the core material is in the form of a multiplicity of nanoparticles.

1 1 . A delivery system as claimed in claim 10, wherein the nanoparticles are of the order of 100 nm in diameter.

12. A delivery system as claimed in claim 10 or claim 1 1 when dependent on claim 2 or claim 4, wherein the nanoparticles are mesoporous silica nanoparticles.

13. A delivery system as claimed in any of claims 1 to 8, wherein the core material comprises a substrate.

14. A delivery system as claimed in any of claims 1 to 8, wherein the core material comprises a part of an article of manufacture.

15. A delivery system as claimed in any preceding claim, wherein the core material is able to be attracted by a magnet.

16. A delivery system as claimed in any of claims 3 to 1 5, wherein the capping material comprises a degradable material.

17. A delivery system as claimed in claim 16, wherein the degradable material is selected such as to be desirable to, and consumable by, a microorganism being targeted.

18. A delivery system as claimed in claim 1 , claim 2 or claim 17, wherein the degradable material is selected such as to be relatively undesirable to, and relatively unconsumed by, microorganisms not being targeted.

19. A delivery system as claimed in claim 16, wherein the degradable material comprises a soluble material.

20. A delivery system as claimed in claim 16, wherein the degradable material is able to be broken down or opened up by the effect of one or more of:

ultrasound, irradiation, light, UV light, ionic change, pH level, temperature, osmotic change.

21 . A delivery system as claimed in any preceding claim, wherein the capping material is biodegradable.

22. A delivery system as claimed in any preceding claim, wherein the capping material comprises a sugar.

23. A delivery system as claimed in claim 22, wherein the capping material comprises lactose, xylose, sucrose or starch.

24. A delivery system as claimed in any preceding claim, further comprising a dye that is arranged to be released from the core material when the capping material is broken down or opened up.

25. A delivery system as claimed in any preceding claim, wherein the biocide comprises an antimicrobial agent.

26. A delivery system as claimed in claim 25, wherein the antimicrobial agent comprises a plant secondary metabolite.

27. A delivery system as claimed in claim 26, wherein the antimicrobial agent comprises allyl isothiocyanate or cinnamaldehyde.

28. A delivery system as claimed in claim 25, wherein the antimicrobial agent is an antibacterial agent.

29. A delivery system as claimed in claim 25, wherein the antimicrobial agent is an antifungal agent.

30. A delivery system as claimed in claim 25, wherein the antimicrobial agent is an antiviral agent.

31 . A method comprising:

identifying a microorganism to be targeted;

identifying a biocide suitable for combating the microorganism to be targeted;

identifying a degradable material that is desirable to, and consumable by, the microorganism to be targeted; and

designing a delivery system for the said biocide, wherein:

the delivery system comprises a core material;

the said biocide is located within or on the core material; and

the delivery system further comprises a capping material comprising the said degradable material, applied to the core material so as to contain the biocide within or on the core material, and to enable exposure of the biocide when the capping material is broken down or opened up.

32. A method as claimed in claim 31 , further comprising manufacturing the delivery system.

33. A method of manufacturing a delivery system for a volatile biocide, the method comprising:

providing a porous core material having a multiplicity of pores;

introducing a volatile biocide into or onto the core material; and

applying a capping material to the core material so as to contain the biocide within or on the core material, such that the biocide is able to be exposed when the capping material is broken down or opened up.

34. A method as claimed in any of claims 31 to 33, wherein the core material is mesoporous.

35. A method as claimed in claim 34, wherein the core material contains pores having a diameter in the range of 2 nm to 50 nm.

36. A method as claimed in claim 35, wherein the pores are of the order of 2 nm in diameter.

37. A method of manufacturing a delivery system for a biocide, the method comprising:

providing a substantially non-porous core material;

introducing a biocide onto the core material such that the biocide is adsorbed onto the surface of the core material; and

applying a capping material to the core material so as to contain the biocide on the core material, such that the biocide is able to be exposed when the capping material is broken down or opened up.

38. A method as claimed in claim 37, wherein the biocide is volatile prior to being adsorbed onto the surface of the core material.

39. A method as claimed in any of claims 31 to 38, wherein the core material is in the form of a multiplicity of discrete particles, such that the delivery system is particulate in form.

40. A method as claimed in claim 39, wherein the core material is in the form of a multiplicity of nanoparticles.

41 . A method as claimed in claim 40, wherein the nanoparticles are of the order of 100 nm in diameter.

42. A method as claimed in claim 40 or claim 41 when dependent on claim 34, wherein the nanoparticles are mesoporous silica nanoparticles.

43. A method as claimed in any of claims 31 to 38, wherein the core material comprises a substrate.

44. A method as claimed in any of claims 31 to 38, wherein the core material comprises a part of an article of manufacture.

45. A method as claimed in any of claims 31 to 44, wherein the core material is able to be attracted by a magnet.

46. A method as claimed in any of claims 33 to 45, wherein the capping material comprises a degradable material.

47. A method as claimed in claim 46, wherein the degradable material is selected such as to be desirable to, and consumable by, a microorganism being targeted.

48. A method as claimed in claim 31 , claim 32 or claim 47, wherein the degradable material is selected such as to be relatively undesirable to, and relatively unconsumed by, microorganisms not being targeted.

49. A method as claimed in claim 46, wherein the degradable material comprises a soluble material.

50. A method as claimed in claim 46, wherein the degradable material is able to be broken down or opened up by the effect of one or more of:

51 . A method as claimed in any of claims 31 to 50, wherein the capping material is biodegradable.

52. A method as claimed in any of claims 31 to 51 , wherein the capping material comprises a sugar.

53. A method as claimed in claim 52, wherein the capping material comprises lactose, xylose, sucrose or starch.

54. A method as claimed in any of claims 32 to 53, further comprising introducing a dye to the core material, the dye being arranged to be released from the core material when the capping material is broken down or opened up.

55. A method as claimed in any of claims 31 to 54, wherein the biocide comprises an antimicrobial agent.

56. A method as claimed in claim 55, wherein the antimicrobial agent comprises a plant secondary metabolite.

57. A method as claimed in claim 56, wherein the antimicrobial agent comprises allyl isothiocyanate or cinnamaldehyde.

58. A method as claimed in claim 55, wherein the antimicrobial agent is an antibacterial agent.

59. A method as claimed in claim 55, wherein the antimicrobial agent is an antifungal agent.

60. A method as claimed in claim 55, wherein the antimicrobial agent is an antiviral agent.

61 . A method of delivering a biocide, the method comprising:

providing a delivery system as claimed in any of claims 1 to 30;

introducing the delivery system to a microorganism or to an environment to be protected from said microorganism; and

allowing the capping material to break down or be opened up, thereby releasing the biocide from the core material.

62. A method as claimed in claim 61 , wherein the delivery system is as claimed in claim 1 or claim 17, such that the microorganism consumes at least some of the capping material and thereby releases the biocide.

63. A method as claimed in claim 61 , wherein the delivery system is as claimed in claim 19, such that the capping material is at least partially soluble in a medium in the vicinity of the microorganism, and the method further comprises introducing the delivery system to the medium such that the capping material breaks down and thereby releases the biocide.

64. A method as claimed in claim 61 , wherein the delivery system is as claimed in claim 20, and the method further comprises breaking down or opening up the capping material by the effect of one or more of:

65. A method as claimed in claim 64, wherein the said effect is automatically triggered by control means in response to receiving an activation signal.

66. A method as claimed in claim 65, wherein the activation signal is provided by a sensor.

67. A method as claimed in claim 66, wherein the sensor is a detector of a particular microorganism.

68. A method as claimed in any of claims 61 to 67, wherein the delivery system is as claimed in claim 15, and the method further comprises attracting the core material using a magnet.

69. A healthcare product comprising a delivery system as claimed in any of claims 1 to 30.

70. A healthcare product as claimed in claim 69 when dependent on claim 28, being an antibacterial handwash or disinfectant.

71 . A healthcare product as claimed in claim 69 when dependent on claim 29, being an antifungal product.

72. A healthcare product as claimed in claim 69 when dependent on claim 30, being an antiviral product.

73. A bio-industrial product comprising a delivery system as claimed in any of claims 1 to 30.

74. A bio-industrial product as claimed in claim 73, being for preventing build-up of unwanted microbial growth in metalworking fluids, water circulation systems, air conditioning systems and such like.

75. A bio-industrial product as claimed in claim 73, being for use in agriculture.

76. A biocide delivery product comprising a delivery system as claimed in any of claims 1 to 30.

77. A delivery system for a biocide substantially as herein described with reference to and as illustrated in any combination of the accompanying drawings.

78. A method of designing a delivery system for a biocide substantially as herein described with reference to and as illustrated in any combination of the accompanying drawings.

79. A method of manufacturing a delivery system for a biocide substantially as herein described with reference to and as illustrated in any combination of the accompanying drawings.

80. A method of delivering a biocide substantially as herein described with reference to and as illustrated in any combination of the accompanying drawings.

81 . A healthcare or bio-industrial product, or other biocide delivery product, substantially as herein described with reference to and as illustrated in any combination of the accompanying drawings.