FIELD OF THE INVENTION

The present invention relates to a composition, a process for producing the composition, and uses of the composition as an antimicrobial agent or as a pharmaceutical for treating microbial infections, tumours, cancer or thrombosis, or for stimulating or enhancing the immune system. The invention further relates to antimicrobial films, coatings, coated products, packaging materials, and polymer compositions, and to processes for producing those products.

BACKGROUND TO THE INVENTION

Microbial resistance to known antimicrobials is a growing problem. There is therefore an ongoing need to develop new antimicrobial agents and compositions.

Since ancient times, garlic has been known to be a natural antibiotic for a broad range of bacteria and fungi. Allicin is the chemical agent attributed to be the active ingredient of the beneficial therapeutic properties of garlic. Allicin is an organic disulfide chemical compound, CH ₂ =CHCH ₂ SS(O)CH ₂ CH=CH ₂ , which is not present in garlic unless tissue damage takes place and activates the Allinase enzyme contained in the garlic cloves to react with Alliin, which then forms Allicin. However, Allinase is irreversibly deactivated under acidic pHs (e.g. internal human body) thus Allicin is generally not produced in the body from the consumption of garlic. Allicin is also unstable when utilized, degrading rapidly with heat and more slowly upon standing at room temperature. The potential antimicrobial and antifungal properties of Allicin are diminished by its instability.

SUMMARY OF THE INVENTION

It is a finding of the present invention that the natural antibiotic properties of Allicin, an active ingredient of garlic, are enhanced when Allicin is incorporated into nanoparticles of an inorganic carrier material, for instance into nanoparticles of a zeolite, a clay, a metal oxide or a metal such as gold. It has been found that such nanoparticles, when functionalized with Allicin, demonstrate significantly improved antimicrobial performance compared to Allicin alone and also when benchmarked against conventional antibiotic compounds. In some cases, the minimum inhibition concentration, MIC, of the Allicin-containing nanoparticles was 50 times lower that that of Allicin alone.

The fiinctionalisation of nanoparticles with Allicin also has the benefit of providing protection and stability to the Allicin, in addition to increasing its antimicrobial reactivity. Indeed, the antibiotic activity of the nanoparticles was shown to be remarkably stable, with significant antimicrobial activity over long periods of storage. The antimicrobial properties of Allicin alone, on the other hand, are lost after just a few days.

Thus, novel Allicin-functionalized nanoparticles have been designed with enormous potential to enhance biocidal action against Gram -positive and -negative bacteria, antibiotic resistant strains (such as MRSA), fungi and pathogenic protozoa. The Allicin-containing nanomaterials also have antithrombotic properties, have the potential to treat cancer or tumours, and can be used to stimulate or enhance the immune system.

Accordingly, the invention provides a composition comprising nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier.

The invention also provides a process for producing a composition comprising nanoparticles, which nanoparticles comprise allicin and an inorganic carrier, the process comprising: (a) treating precursor nanoparticles, comprising said inorganic carrier, with Allicin.

Usually, the process of the invention further comprises: (b) recovering said composition.

In another aspect, the invention provides a composition which is obtainable by the process of the invention.

The invention also provides:

- a pharmaceutical composition comprising nanoparticles and a pharmaceutically acceptable carrier or diluent, which nanoparticles comprise Allicin and an inorganic carrier;

- a composition comprising nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier, for use in a method for treatment of the human or animal body by therapy;

- an antimicrobial composition comprising nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier;

- a composition comprising nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier, for use in the treatment of a bacterial infection, a fungal infection, a viral infection, an infection by a pathogenic protozoan, a tumour, cancer or thrombosis, or for use in a method of stimulating or enhancing the immune system; - use of a composition comprising nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier, in the manufacture of a medicament for use in the treatment of a bacterial infection, a fungal infection, a viral infection, an infection by a pathogenic protozoan, a tumour, cancer or thrombosis, or in a method of stimulating or enhancing the immune system;

- a method of treating a bacterial infection, a fungal infection, a viral infection, a parasitic infection, a tumour, cancer or thrombosis, or a method of stimulating or enhancing the immune system, which method comprises administering to a patient in need of such treatment an effective amount of a composition comprising nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier;

- an agent for the treatment of a bacterial infection, a fungal infection, a viral infection, an infection by a pathogenic protozoan, a tumour, cancer or thrombosis, or for stimulating or enhancing the immune system, comprising a composition which comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier;

- a spermicidal composition comprising a spermicide and nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier;

- a film which comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier;

- a coating which comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier;

- a coated product, which comprises a product and a coating disposed on a surface thereof, wherein the coating comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier;

- a packaging material, which packaging material comprises a polymer and nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier;

- a polymer composition, which polymer composition comprises a polymer and nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier;

- a process for producing a coating, which coating comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier, which process comprises: disposing a coating composition which comprises nanoparticles and a solvent onto a substrate, which nanoparticles comprise Allicin and an inorganic carrier; and removing said solvent; - a process for producing a film, which film comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier, which process comprises: disposing a film-forming composition which comprises nanoparticles and a solvent onto a substrate or mould, which nanoparticles comprise Allicin and an inorganic carrier; and removing said solvent.

- a process for producing a coated product, which coated product comprises a product and a coating disposed on a surface thereof, wherein the coating comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier, which process comprises: disposing a coating composition which comprises nanoparticles and a solvent onto a product to be coated, which nanoparticles comprise Allicin and an inorganic carrier; and removing said solvent.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a schematic illustration of the production of Allicin from Alliin using the Allinase enzyme.

Fig. 2 shows a schematic illustration of the reduction of [AuCl ₄ ] ^" to form gold nanoparticles in the presence of Allicin, to produce Allicin-functionalised gold nanoparticles in accordance with the invention.

Fig. 3 shows a schematic illustration of the ion-exchange reaction between Allicin and zeolite nanoparticles, to produce Allicin-functionalised zeolite nanoparticles in accordance with the invention.

Fig. 4 shows the zeta potential distribution of a composition comprising Allicin- functionalised gold nanoparticles in accordance with the invention.

Fig. 5 shows the zeta potential distribution of Allicin-functionalised zeolite nanoparticles in accordance with the invention.

Fig. 6 shows the zeta potential distribution of an extract of Allicin.

Fig. 7 shows the zeta potential distribution of zeolite.

Fig. 8 shows the zeta potential distribution of gold nanoparticles.

Fig. 9 shows the x-ray photoelectron spectrum of a composition comprising Allicin- functionalised gold nanoparticles in accordance with the present invention. The spectrum shows the presence of the distinctive sulphur peaks of Allicin as well as the distinctive Au peaks of the gold present in the nanoparticles.

Fig. 10 is an electron micrograph of gold nanoparticles functionalised with Allicin in accordance with the invention. The micrograph shows that the average particle size of the gold nanoparticles is under 20 nm. The nanoparticles thereby provide a high surface area of contact with Allicin.

Figs. 11 and 12 are electron micrographs of gold nanoparticles functionalised with Allicin in accordance with the invention. The figures confirm that the particles have a low tendency to agglomerate, indicating that the individual particles have a high stability. The micrographs also show that the nanoparticles have a uniform shape and size.

Fig. 13 is a bar chart summarising the results of antibiotic disk diffusion tests on Gram negative bacteria, the left hand bar representing a composition comprising Allicin/gold nanoparticles at a concentration of 4 μg/mL, the right hand bar representing a composition comprising Allicin/zeolite nanoparticles at the same concentration and the y axis representing the inhibition of E. coli in terms of the inhibition diameter in units of millimeters. The horizontal bar represents the minimum inhibition concentration for a composition comprising Allicin at a concentration of 210 μg/mL.

Fig. 14 is a bar chart showing the results of antibiotic disk diffusion tests on Gram positive bacteria, in which the left hand bar represents a composition comprising gold/Allicin nanoparticles at a concentration of 4 μg/mL, the right hand bar represents a composition comprising zeolite/ Allicin nanoparticles at the same concentration, and the y axis represents inhibition of B. subtilis in terms of the inhibition diameter in units of millimeters.

Fig. 15 is a bar chart summarising the results of antibotic disk diffusion tests on Gram negative bacteria after storing gold/Allicin and zeolite/ Allicin compositions of the invention at 4°C for 30 days. The left hand bar represents a composition comprising gold/Allicin nanoparticles at a concentration of 2 μg/mL, the right hand bar represents a composition comprising zeolite/Allicin nanoparticles at a concentration of 2 μg/mL, both compositions having been stored at 4°C for 30 days, and the y axis represents inhibition of E. coli in terms of inhibition diameter in units of millimeters.

Fig. 16 is a bar chart summarising the results of antibiotic disk diffusion tests on Gram positive bacteria over time, the nanoparticle compositions of the invention having been stored at 4°C for a period of 30 days. The left hand bar represents a composition comprising Allicin/gold nanoparticles at a concentration of 2 μg/mL, the right hand bar represents a composition comprising Allicin/zeolite nanoparticles at the same concentration, both compositions having been stored for 30 days at 4°C, and the y axis represents inhibition of B. subtilis in terms of inhibition diameter in units of millimeters.

Fig. 17 is a graph of growth of the bacterial strain E. coli in terms of absorption units (A.U.) at a wavelength of 600nm (y axis) versus time in units of hours (x axis) both in the presence (lower plot) and absence (upper plot) of a composition of gold/Allicin nanoparticles in accordance with the invention.

Fig. 18 is a graph of growth of the bacterial strain B. subtilis in trems of absorption units (A.U.) at a wavelength of 600nm (y axis) versus time in units of hours (x axis) both in the presence (lower plot) and absence (upper plot) of a composition of gold/Allicin nanoparticles in accordance with the invention.

Fig. 19 is a graph of growth of the bacterial strain E. coli in terms of absorption units (A.U.) at a wavelength of 600nm (y axis) versus time in units of hours (x axis) both in the presence (lower plot) and absence (upper plot) of a composition comprising zeolite/Allicin nanoparticles in accordance with the invention.

Fig. 20 is a graph of growth of the bacterial strain B, subtilis in terms of absorption units (A.U.) at a wavelength of 600nm (y axis) versus time in units of hours (x axis) both in the presence (lower plot) and absence (upper plot) of a composition comprising zeolite/Allicin nanoparticles in accordance with the invention.

Figs. 21 (a) and (b) are TEM images of healthy E. coli bacteria, before treatment with a gold/Allicin or a zeolite/Allicin nanoparticle composition of the invention.

Figs. 22 (a) to (d) are TEM images of E. coli bacteria after treatment with a gold/Allicin or a zeolite/Allicin nanoparticle composition of the invention.

Fig. 23 is a bar chart showing the percentages of alive and dead E. coli bacteria (y axis) both immediately after contact (left-hand pair of bars) and at 30 minutes after contact (right-hand pair of bars) with a gold/Allicin nanoparticle composition of the invention. The left-hand bar in each pair shows the percentage of live bacteria whereas the right-hand bar in each pair shows the percentage of dead bacteria.

Fig. 24 is a bar chart showing the percentages of alive and dead E. coli bacteria (y axis) both immediately after contact (left-hand pair of bars) and at 30 minutes after contact (right-hand pair of bars) with a zeolite/Allicin nanoparticle composition of the invention. The left-hand bar in each pair shows the percentage of live bacteria whereas the right-hand bar in each pair shows the percentage of dead bacteria.

Fig. 25 is a bar chart showing the diameter of the bacterial inhibition, in units of mm, caused by perfluoroalkoxy (PFA) polymer films comprising Au-Allicin nanoparticles (y axis), on gram-negative E. coli (middle bar), gram-positive B. subtilis (right hand bar) and control (left hand bar).

DETAILED DESCRIPTION OF THE INVENTION

The composition of the invention comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier.

As used herein, the term "nanoparticle" means a microscopic particle whose size is measured in nanometres (run). Typically, a nanoparticle has particle size of from 0.1 nm to 1000 nm. More typically, a nanoparticle has a particle size of from 0.5 nm to 1000 nm, from 1 nm to 1000 nm, or for instance from 1 nm to 800 nm or from 1 nm to 600 nm. A nanoparticle may be crystalline or amorphous. It may be spherical or non-spherical. Non- spherical nanoparticles may for instance be plate-shaped, needle-shaped or tubular. The term "particle size" as used herein means the diameter of the particle if the particle is spherical or, if the particle is non-spherical, the volume-based particle size. The volume- based particle size is the diameter of the sphere that has same volume as the non-spherical particle in question.

The nanoparticles in the composition of the invention comprise Allicin and an inorganic carrier. Any suitable inorganic carrier may be used, provided that the inorganic carrier can exist in nanoparticle form. Typically, the inorganic carrier comprises a metal, a metal oxide, a zeolite or a clay. The Allicin may be attached to or associated with the inorganic carrier through a physical interaction between the inorganic carrier and Allicin (e.g. due to an electrostatic attraction between the inorganic carrier and Allicin) and/or through a mechanical interaction between the inorganic carrier and Allicin (e.g. due to Allicin being trapped within a porous structure of the inorganic carrier) or through the formation of one or more chemical bonds between the inorganic carrier and Allicin (e.g. covalent bonds, ionic bonds, hydrogen bonds or other non-covalent bonds). Thus Allicin may be covalently or non-covalently bonded to the inorganic carrier or, for instance, physically or chemically adsorbed onto the surface of the inorganic carrier or physically or chemically held within the structure of the inorganic carrier. Without wishing to be bound by theory, it is thought that the nanoparticles of the composition of the invention have increased antimicrobial and pharmaceutical effects compared to Allicin alone because the nanoparticles present a high surface area of Allicin and also because Allicin is stabilised in the presence of the inorganic carrier. In one embodiment, the composition consists of said nanoparticles. In this embodiment, the nanoparticles are the sole component of the composition. In another embodiment, the composition consists essentially of said nanoparticles. In this embodiment other components may be present, for instance a solvent or carrier, such as a pharmaceutically acceptable carrier, or for instance an impurity, such as a starting material that was used to produce the composition, or a by-product, provided that the essential characteristics of the composition are not materially affected by their presence. In yet another embodiment, the composition includes one or more further components in addition to the nanoparticles, for instance one or more solvents, starting materials, impurities, byproducts or one or more further active agents that enhance or modify the effect of the composition as an antibacterial agent or pharmaceutical.

Typically, the nanoparticles in the composition of the invention have a mean particle size of less than or equal to 800 nm. More typically, the mean particle size is less than or equal to 600 nm or, for instance, less than or equal to 400 nm. Even more typically, the mean particle size is less than or equal to 200 nm, for instance less than or equal to 150 nm, less than or equal to 120 nm, or less than or equal to 100 nm. In one embodiment, the mean particle size of the nanoparticles is from 0.1 to 150 nm, from 0.5 to 150 nm, or from 20 to 150 nm, more typically from 40 to 120 nm, even more typically from 60 to 100 nm. In another embodiment, the mean particle size of the nanoparticles in the composition of the invention is less than or equal to 80 nm, for instance less than or equal to 40 nm or less than or equal to 25 nm. In one embodiment, the mean particle size is less than or equal to 20 nm, or less than or equal to 10 nm. In another embodiment, the mean particle size is less than or equal to 5 nm, for instance less than or equal to 2 nm. The mean particle size may for instance be from 0.1 nm to 25 nm, to 20 nm, to 10 nm, to 5nm or to 2nm, or for instance from 0.5 nm to 25 nm, to 20 nm, to 10 nm, to 5nm or to 2nm.

Typically, the nanoparticles of the composition of the invention have a zeta potential which is less (i.e. more negative) than that of Allicin. Typically, Allicin has a zeta potential of about -6.5 mV. Thus, the nanoparticles which comprise Allicin and the inorganic carrier typically have a zeta potential which is equal to or less than -7.0 mV. This reflects the stability of the nanoparticles when compared to Allicin alone; colloids of nanoparticles having a zeta potential of a higher magnitude (in this case, a more negative zeta potential) are more electrically stabilized compared to colloids of nanoparticles with a lower zeta potential. Particles having a zeta potential of a lower magnitude tend to coagulate or flocculate.

Usually, the nanoparticles of the composition of the invention have a zeta potential of less than or equal to -8.0 mV, for instance less than or equal to to -8.5 mV. hi another embodiment, the nanoparticles have a zeta potential of less than or equal to -10 mV, less than or equal to -15 mV, or less than or equal to -20 mV.

The inorganic carrier typically comprises a metal (usually an elemental metal, having an oxidation state of zero), a metal oxide, a zeolite or a clay. More typically, the inorganic carrier comprises a metal or a zeolite.

Typically, the inorganic carrier is non-toxic. Usually, it is fairly inert, and typically it does not readily oxidise in the presence of air. Thus, when the inorganic carrier is a metal, gold is particularly suitable.

Typically, the inorganic carrier does not itself have any antimicrobial effect when in nanoparticle form. As demonstrated in the control experiments in the Examples, gold and zeolite nanoparticles do not show any antimicrobial effects in the absence of Allicin.

Thus, when the inorganic carrier is a metal it is typically gold.

Metal oxides that can suitably be employed as the inorganic carrier in the composition of the invention include zinc oxide and titanium oxide.

In one embodiment, the inorganic carrier comprises a zeolite, i.e. an aluminosilicate. Some of the more common mineral zeolites, which may be used as the inorganic carrier in the compositions of the present invention, include analcime, chabazite, heulandite, natrolite, phillipsite, and stilbite. Zeolites have a porous aluminosilicate structure that can accommodate a wide variety of cations, such as Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ and others. These positive ions are fairly loosely held within the aluminosilicate framework of the zeolite and can readily be exchanged for others in a contact solution. Nanoparticles for the composition of the present invention can be prepared by exchanging Allicin with the cations of a zeolite, in an ion-exchange reaction.

In one embodioment, the inorganic carrier comprises a zeolite selected from, or derived from, any of the following zeolites: amicite, analcime, barrerite, bellbergite, bikitaite, boggsite, brewsterite, chabazite, clinoptilolite, cowlesite, dachiardite, edingtonite, epistilbite, erionite, faujasite, ferrierite, garronite, gismondine, gmelinite, gobbinsite, gonnardite, goosecreekite, harmotome, herschelite, heulandite, laumontite, levyne, maricopaite, mazzite, merlinoite, mesolite, montesommaite, mordenite, natrolite, offretite, paranatrolite, paulingite, pentasil, perlialite, phillipsite, pollucite, scolecite, sodalite, sodium dachiardite, stellerite, stilbite, tetranatrolite, thomsonite, tschemichite, wairakite, wellsite, willhendersonite and yugawaralite. By "derived from", in this context, is meant that the inorganic carrier may comprise a zeolite whose structure is the same as one of the zeolites listed above except that the cation or cations of that zeolite (typically Na ⁺ , K ⁺ , Ca ²⁺ and/or Mg ²⁺ ) have been partially or wholly replaced by Allicin. hi one embodiment, the inorganic carrier comprises a zeolite which is, or is derived from [K ₆ Na ₃ (H ₂ O) ₂₁ ][Al ₉ Si ₂₇ O ₇₂ ]. Nanocrystalline zeolites which may be employed as the inorganic carrier in the present invention are commercially available, for instance from NanoScape in Germany.

In one embodiment, the inorganic carrier is a clay. Particularly suitable clays, which can be functionalised with Allicin to provide a composition of the invention, include those which are available in nanoparticle form ("nanoclays"). Such nanoclays are commercially available, and include the bentonites and montmorillonite. Typically, the mean particle size of such nanoclays is less than or equal to 200 run.

In one embodiment, the inorganic carrier comprises gold or a zeolite.

When the inorganic carrier comprises gold, the mean particle size of the nanoparticles in the composition of the invention is usually less than or equal to 25 nm, more typically less than or equal to 20 nm, or less than or equal to 10 nm. The mean particle size may for instance be from 0.1 nm to 25 nm, to 20 nm, to 10 nm, to 5nm or to 2nm, or for instance from 0.5 nm to 25 nm, to 20 nm, to 10 nm, to 5nm or to 2nm. Such small partical sizes are beneficial because they facilitate interaction of the nanoparticles with target microbial cells such as bacteria, thereby increasing the effectiveness of the nanoparticles as antimicrobial agents, hi one embodiment, the mean particle size is less than or equal to 5 nm, for instance less than or equal to 2 nm.

Usually, when the inorganic carrier comprises gold, the nanoparticles have a zeta potential of less than or equal to -8.0 mV, or for instance, less than or equal to -8.5 mV. A small particle size in combination with a high-magnitude zeta potential indicates that the nanoparticles not only provide a high surface area of active ingredient (Allicin) but also are stable, with little tendancy to coagulate or flocculate.

When the inorganic carrier comprises a zeolite, the mean particle size of the nanoparticles is typically less than or equal to 150 nm, more typically less than or equal to 120 nm or less than or equal to 100 nm. In one embodiment, the inorganic carrier comprises a zeolite and the mean particle size of the nanoparticles is from 20 to 150 nm, more typically from 40 to 120 nm, even more typically from 60 to 100 nm.

Typically, when the inorganic carrier comprises a zeolite, the nanoparticles have a zeta potential of less than or equal to -10 mV, more typically less than or equal to -15 mV, or less than or equal to -20 mV.

Typically, the composition of the invention further comprises a solvent. The nanoparticles are typically present as a suspension or as a dispersion in the solvent, but may also be present in solution. In one embodiment, the nanoparticles are present as a colloidal suspension.

Any suitable solvent may be used. Typically, the solvent is a polar protic solvent. More typically, the solvent comprises water. Optionally, one or more further solvents may be present. For instance, the solvent may comprise a mixture of a polar organic solvent, for instance an alcohol, and water. Typically, the alcohol is a primary alcohol, for instance ethanol. Usually the solvent comprises water or a mixture of water and ethanol. When the solvent is a mixture of water and ethanol, typically up to 20 % v/v ethanol is present. When a solvent is present, the composition of the invention may further comprise a buffer, for instance a sodium phosphate buffer. In one embodiment the solvent is water and the composition further comprises a buffer. In another embodiment, the solvent is a mixture of water and ethanol and the composition further comprises a buffer. Typically, the buffer is a sodium phosphate buffer.

When the composition of the invention comprises a solvent, the concentration of Allicin in the composition may be chosen to suit the application for which the dispersion or suspension of nanoparticles is intended. In one embodiment, the concentration of Allicin in the composition is at least 1 μg/ml, for instance at least 2 μg/ml or, for instance, at least 4 μg/ml. Thus, the concentration of Allicin may be from about 1 to about 50 μg/ml or, for instance, from about 2 to about 50 μg/ml or about 4 to 50 μg/ml. In one embodiment, the concentration of Allicin is from about 1 to about 10 μg/ml, for instance from about 2 to about 10 μg/ml or from about 2 to about 8 μg/ml. Such concentrations of Allicin, when the Allicin is part of a nanoparticle together with an inorganic carrier, are effective against bacteria; they are also at least 50 times lower than the concentrations of Allicin required to achieve the equivalent antibacterial effect when Allicin is not associated with a nanoparticle. The concentration of Allicin may be measured using a known spectrophotometric assay for Allicin which employs the chromogenic thiol 4- Mercaptopyridine (Miron T. Anal Biochem. 2002, 307 (1), 76-83).

Suspensions and solutions of the Allicin-containing nanoparticles can be stored at below room temperature, for instance at about 4°C, for longer lasting efficacy but they can also be stored at room temperature.

In one embodiment, the composition of the invention is a solid composition. In this embodiment, the nanoparticles are present in the solid state as opposed to being suspended or dissolved in a solvent. In the solid state, the nanoparticles can easily be stored and shipped. If required, the solid composition can later be suspended (or dissolved) in a solvent or carrier for use.

Such solid state compositions of the invention may be produced directly from the starting materials (i.e. by treating precursor nanoparticles comprising said inorganic carrier with Allicin). More typically, though, a solid state composition of the invention is obtained by removing solvent from a suspension or solution of the nanoparticles. The solvent may be removed using any suitable technique, for instance by evaporation or by freeze-drying. In one embodiment, the composition is freeze-dried.

The process of the invention, for producing a composition comprising nanoparticles, which nanoparticles comprise allicin and an inorganic carrier, comprises: (a) treating precursor nanoparticles, comprising said inorganic carrier, with Allicin.

The reaction, i.e. step (a), may be carried out in the presence or absence of a solvent, but is usually carried out in the presence of a solvent. Typically, therefore, the reaction mixture comprises the product nanoparticles (comprising Allicin and an inorganic carrier) and a solvent. The reaction mixture may also comprise one or more unreacted starting materials, e.g. unreacted precursor nanoparticles or unreacted free Allicin. The reaction mixture may also contain by-products (i.e. reaction products other than the nanoparticles comprising Allicin and an inorganic carrier) and/or impurities.

When a solvent is present, the solvent is usually a polar protic solvent. More typically, the solvent comprises water. Optionally, one or more further solvents may be present. For instance, the solvent may comprise a mixture of a polar organic solvent, for instance an alcohol, and water. Typically, the alcohol is a primary alcohol, for instance ethanol. Usually the solvent comprises water or a mixture of water and ethanol. When the solvent is a mixture of water and ethanol, typically up to 20 % v/v ethanol is present. When a solvent is present, a buffer may also be present, for instance a sodium phosphate buffer.

In one embodiment, step (a) of treating the precursor nanoparticles with Allicin yields the composition of the invention directly. Thus, for instance, any solvent, if present, and/or any further components present in the reaction mixture may be retained in the composition of the invention.

In another embodiment, the composition is recovered from the reaction mixture, either as a suspension or solution of the nanoparticles, or as a solid composition comprising the nanoparticles. Thus, in one embodiment, the process of the invention further comprises: (b) recovering said composition.

Step (b) may comprise separation of the product nanoparticles (comprising Allicin and the inorganic carrier) from a solvent. Thus, the process of the invention may comprise: (b) recovering said composition, wherein step (b) comprises a solvent-removal step to produce a solid composition.

By removing the solvent to form a solid composition, the composition of the invention material can be easily stored or shipped. The solid composition may later be reconstituted to its original form, if need be, by re-suspending or re-dissolving it in a solvent. Accordingly, in one embodiment, the process further comprises: (c) adding a solvent to the solid composition.

When step (b) comprises a solvent-removal step, the solvent may be removed by any suitable method, including by evaporation or by freeze-drying. If a solid composition is to be produced, then a freeze-drying solvent-removal process is advantageous because it is more likely to keep the Allicin concentration in the nanoparticles undisturbed. Thus, in one embodiment, the solvent removal step comprises a freeze-drying step, to produce a freeze-dried composition.

As an alternative to, or in addition to, a solvent removal step, step (b) may comprise separating the composition of the invention from an unreacted starting material, a byproduct or an impurity.

The composition may be recovered as a suspension or solution of the nanoparticles. Thus, in one embodiment, the process of the invention further comprises: (b) recovering said composition, wherein step (b) comprises isolating a suspension or solution of said nanoparticles, the nanoparticles comprising Allicin and said inorganic carrier, in a solvent. Step (b) may comprise one or more filtration and/or centrifugation steps in order to produce a purified suspension or solution of the product nanoparticles. Thus, in one embodiment, step (b) comprises filtering and/or centrifuging the reaction mixture to produce a purified suspension or solution of said nanoparticles in said solvent.

Usually, the Allicin used in step (a) of the process of the invention is provided in the form of a solution. Typically, the solvent comprises a polar protic solvent, for instance water. More typically, the solvent comprises a mixture of a polar organic solvent and water, for instance a mixture of an alcohol, such as ethanol, and water. When the solvent is a mixture of water and ethanol, typically up to 20 % v/v ethanol is present. A buffer may also be present in the solution, for instance a sodium phosphate buffer. Typically, the concentration of Allicin in the solution is at least 1 μg/ml, for instance at least 2 μg/ml or, for instance, at least 4 μg/ml. Thus, the concentration of Allicin in the solution may be from about 1 to about 50 μg/ml or, for instance, from about 2 to about 50 μg/ml or about 4 to 50 μg/ml. In one embodiment, the concentration of Allicin is from about 1 to about 20 μg/ml, for instance from about 2 to about 15 μg/ml or from about 2 to about 10 μg/ml.

Typically, the Allicin used in the process of the invention is extracted from crushed garlic into the solvent. The resulting extract is typically subjected to a solvent-liquid separation step, for instance a filtration or centrifugation step, or both. Further purification by HPLC may also be performed. The Allicin extract can then either be stored or used immediately in the process of the invention.

Typically, Allicin is used in the process of the invention in a molar excess.

The precursor nanoparticles used in step (a) of the process may themselves be produced using known techniques or may be obtained from a commercial source. For instance, nanocrystalline zeolites, which may be employed as the precursor nanoparticles in the process of the invention, are commercially available from NanoScape in Germany. Nanoclays are also commercially available, from sources including Sigma Aldrich.

When the inorganic carrier is a metal, for instance gold, the precursor nanoparticles are typically produced by reducing a salt of the metal, in solution, to produce a colloidal suspension or solution of the metal which is then ready for reaction with Allicin to form the nanoparticles of the composition of the invention. The reduction of the metal salt may be effected chemically, by treating the solution with a reducing agent, or electrochemically, by submitting the solution to electrolysis. In the case of gold, a solution of a gold halide salt (for instance chloroauric acid) may be treated with a reducing agent (for instance sodium citrate) to produce a suspension of colloidal gold nanoparticles. Such a suspension typically has a purple/red wine colour.

Usually, a solvent is present during the reaction, thus step (a) typically comprises treating a suspension or solution of the precursor nanoparticles in a solvent with Allicin.

Typically, the precursor nanoparticles have a zeta potential which is less (i.e. more negative) than that of Allicin. Thus, the precursor nanoparticles typically have a zeta potential which is equal to or less than -7.0 mV. More typically, the precursor nanoparticles have a zeta potential of less than or equal to -10.0 mV, less than or equal to - 15.0 mV, or less than or equal to -20.0 mV. Even more typically, the precursor nanoparticles have a zeta potential of less than or equal to -25.0 raV or less than or equal to -30.0 mV. This facilitates the reaction of the precursor nanoparticles with Allicin in order to produce nanoparticles, which comprise Allicin and the inorganic carrier, that themselves have a zeta potential which is less (i.e. more negative) than that of Allicin. This is advantageous because colloids of nanoparticles which have a high magnitude zeta potential are more electrically stable, whereas those having a lower (magnitude) zeta potential tend to coagulate or flocculate in suspension.

When a solvent is present during the reaction, and step (a) comprises treating a suspension or solution of the precursor nanoparticles with Allicin, the suspension or solution may be obtained by first suspending or dissolving the precursor nanoparticles in a suitable solvent. Thus, in one embodiment, step (a) comprises suspending or dissolving the precursor nanoparticles in a solvent and treating the suspension or solution with Allicin. Typically, when the inorganic carrier comprises a zeolite, nanoclay or metal oxide, step (a) comprises suspending the precursor nanoparticles in a suitable solvent and treating the suspension with Allicin.

Alternatively, step (a) may comprise forming a suspension or solution of the precursor nanoparticles in the presence of Allicin. In this embodiment, Allicin is present when the precursor nanoparticles are formed as a suspension or solution, allowing Allicin immediately to react or otherwise associate with the precursor nanoparticles, to produce nanoparticles which comprise both Allicin and the inorganic carrier.

When the inorganic carrier comprises a metal, for instance gold, step (a) usually comprises forming a suspension or solution of the precursor nanoparticles in said solvent in the presence of Allicin. More typically, step (a) comprises forming a suspension of the precursor nanoparticles in said solvent in the presence of Allicin. Typically, the suspension of the precursor nanoparticles is a colloidal suspension of the metal.

The suspension or solution of the precursor metal nanoparticles is usually formed by treating a solution comprising a salt of the metal with a reducing agent. Thus, when the inorganic carrier comprises a metal, step (a) usually comprises treating a solution of a salt of a metal with a reducing agent.

Any suitable reducing agent may be used. Citrate may be used as the reducing agent, for instance sodium citrate. Allicin itself can also be used as the reducing agent: Allicin is itself capable of reducing the metal ions in solution to form a colloidal suspension of the precursor nanoparticles, which can then react with the Allicin to produce the nanoparticles of the invention that comprise both Allicin and the inorganic carrier. Thus, in one embodiment, step (a) comprises treating a solution of a salt of a metal with Allicin, wherein Allicin reduces the metal in said salt to form a colloidal suspension of the metal in the presence of Allicin.

Thus, in one embodiment, the inorganic carrier is a metal and the process of the invention comprises: (a) treating a solution of a metal salt with Allicin.

More typically, Allicin is used in combination with a further reducing agent. Any suitable reducing agent may be used as the further reducing agent, although typically citrate is used, for instance sodium citrate. Thus in one embodiment, step (a) comprises: treating a solution comprising a salt of said metal with Allicin and a further reducing agent, thereby forming said colloidal suspension of said metal in the presence of Allicin.

Any suitable soluble metal salt can be used. Usually, when the metal is gold, a gold halide salt is used, for instance chloroauric acid, H[AuCl ₄ ].

Step (a) may be carried out in the presence of heat. Thus, step (a) may comprise heating the reaction mixture to a temperature equal to or greater than 50 ⁰ C, for instance equal to or greater than 80 ⁰ C, or equal to or greater than 100 ⁰ C.

In one embodiment, the inorganic carrier is gold and the process of the invention comprises: (a) treating a solution of a gold salt with Allicin and citrate, in the presence of heat. Typically, the gold salt is chloroauric acid. Typically, the reaction is carried out under agitation, e.g. stirring. Usually, the process further comprises (b) filtering and/or centrifuging the reaction mixture to produce a suspension or solution of nanoparticles comprising Allicin and gold. The resulting suspension or solution can be stored below room temperature, e.g. at around 4 ⁰ C, for longer lasting antimicrobial efficacy, but can also be stored at room temperature.

In another embodiment, the nanoparticles comprising Allicin and the inorganic carrier are produced by an ion-exchange reaction between the inorganic carrier and Allicin. This embodiment is particularly effective when the inorganic carrier is a zeolite or a clay, and especially when it is a zeolite. Thus, in one embodiment, step (a) comprises submitting the precursor nanoparticles to an ion exchange reaction with Allicin.

Typically, a solvent is present and the precursor nanoparticles are present as a suspension in the solvent. Thus, in this embodiment, step (a) usually comprises submitting a suspension of the precursor nanoparticles in a solvent to an ion exchange reaction with Allicin. The solvent may be any suitable solvent, but it typically comprises water and may additionally comprise a polar organic solvent, for instance an alcohol, e.g. ethanol. A buffer, for instance sodium phosphate, may also be present.

The ion exchange process is typically stimulated by adding an acid, at a low concentration in the solvent. Thus, step (a) usually comprises treating the suspension of the precursor nanoparticles with Allicin in the presence of an acid. Any suitable acid may be employed, for instance a mineral acid, e.g. hydrochloric acid.

Homogenising the reaction mixture facilitates ion exchance. Thus, step (a) usually comprises treating the suspension of the precursor nanoparticles with Allicin in the presence of an acid and homogenising the reaction mixture. Typically, the reaction mixture is homogenised in a mixer at a speed of from 60 to 100 rpm. Typically, the reaction mixture is homogenised for a period of up to 24 hours, for instance from 2 to 24 hours, more typically from 4 to 24 hours, and even more typically from 12 to 24 hours or from 18 to 24 hours.

Typically, the ion exchange reaction is carried out at room temperature. The concentration of Allicin in the ion-exchange reaction mixture may be at least 1 μg/ml, for instance at least 2 μg/ml or, for instance, at least 4 μg/ml. hi one embodiment, the concentration of Allicin is from about 1 to about 20 μg/ml, for instance from about 2 to about 15 μg/ml or from about 2 to about 10 μg/ml.

In one embodiment, the inorganic carrier is a zeolite and the process of the invention comprises: (a) submitting a suspension of precursor nanoparticles comprising said zeolite in a solvent to an ion exchange reaction, by treating the suspension with Allicin in the presence of an acid and homogenising the reaction mixture, thereby producing said composition comprising nanoparticles, which nanoparticles comprise Allicin and said inorganic carrier.

Uses of the compositions of the invention

It has been found that compositions of the invention demonstrate significantly improved antimicrobial performance compared to Allicin alone and also when benchmarked against conventional antibiotic compounds. Furthermore, the functionalisation of nanoparticles with Allicin provides protection and stability to the active agent, Allicin; the antimicrobial activity of the nanoparticles was shown to be remarkably stable over long periods of time. Thus, the compositions of the invention are useful as antimicrobial agents, in particular as antibacterial agents, antifungal agents, antiviral agents and antiparasitic agents, in both medical and non-medical applications.

The compositions of the invention are particularly useful as therapeutic agents for the treatment or prevention of a microbial infection or disease. The microbial infection or disease may be an infection or disease caused by a bacterium, a fungus, a virus or a parasite, for instance by a pathogenic protozoan.

Bacterial infections or diseases that can be treated or prevented by the compositions of the present invention are those caused by bacteria including, but not limited to, bacteria that have an intracellular stage in its life cycle, such as mycobacteria (e.g. Mycobacteria tuberculosis, M. bovis, M. avium, M. marinum, M. leprae, or M. africanum), rickettsia, mycoplasma, chlamydia, and legionella; Gram positive bacillus (e.g. Listeria, Bacillus subtilis, Bacillus such as Bacillus anthracis, Erysipelothrix species); Gram negative bacillus (e.g. Bartonella, Brucella, Campylobacter, Enterobacter, Escherichia, Francisella, Hemophilus, Klebsiella, Morganella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia species); spirochete bacteria (e.g. Borrelia species including Borrelia burgdorferi that causes Lyme disease); anaerobic bacteria (e.g. Actinomyces and Clostridium species); Gram positive and negative coccal bacteria, Enterococcus species, Streptococcus species, Rhodococcus species, Pneumococcus species, Staphylococcus species, Neisseria species; Arthrobacter species; Flavobacterium species; and Pseudonmonas species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus viridans, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, Escherichia coli and Actinomyces israelii.

Compositions of the invention were shown to have an antibacterial effect on both Gram positive and Gram negative bacteria. The strains tested, for which the compositions of the invention exhibited a remarkably low MIC, included but are not limited to: Staphylococcus sp. (Gram positive), Arthrobacter sp. (Gram positive), Flavobacterium sp. (Gram negative), Rhodococcus sp. (Gram positive), Pseudomonas sp. (Gram negative), E. coli (Gram negative), and Bacillus sp. (Gram positive). Infections or diseases caused by the aforementioned bacteria can therefore be treated or prevented by the compositions of the invention.

The compositions of the invention are also particularly suitable for treating bacteria with resistance to one or more antibiotics. Allicin-containing garlic extract is known to be able to lower the pathogenicity of bacteria by inhibiting the "quorum-sensing" (QS) systems of the bacteria which coordinate virulence and biofilm development (Rasmussen et al., J. Bacteriology, Mar. 2005, p. 1799-1814). Thus, the compositions of the invention may be suitable for down-regulating the expression of virulence factors in bacteria and making biofilms generated by antibiotic-resistant bacteria more susceptible to conventional antibiotic treatments. The compositions of the invention can therefore be used to treat, prevent or ameliorate infections or diseases caused by antibiotic-resistant bacteria or to decrease the antibiotic resistance of such bacteria. In particular, the compositions of the invention may be used to treat MRSA, as evidenced by the fact that they exhibit a low MIC for Staphylococcus bacteria.

Allicin also has antimicrobial activity against mycobacterium species (Hassan N. Biochemical and Biophysical Research Communications, 355 (2007) 471-476; Deshpande, R.G., Journal of Antimicrobial Chemotherapy, 32 (1993) 623-626). Thus, the compositions of the invention are particularly useful as anti-mycobacterium agents, in both medical and non-medical applications, and for treating infections and diseases caused by Mycobacteria. In particular, the compositions of the invention are useful against M. tuberculosis, M. avium and M. marinum.

Compositions of the invention were shown to have an antifungal effect. The fungi strains tested, for which the compositions of the invention exhibited a remarkably low MIC, included Aspergillus sp. including Aspergillus niger and Aspergillus terreus. Other important fungal species that are susceptible to Allicin include Candida albicans. Accordingly, the compositions of the invention are particularly useful as antifungal agents, in both medical and non-medical applications and for treating fungal infections and diseases.

Thus, fungal infections, or conditions resulting from or associated with a fungal infection (e.g., a respiratory infection), can be prevented, treated, managed, and/or ameliorated with the compositions of the invention. Examples of fungi which cause such fungal infections include, but are not limited to, Absidia species (e.g. Absidia corymbifera and Absidia ramosa), Aspergillus species, (e.g. Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger and Aspergillus terreus), Basidiobolus ranarum, Blastomyces dermatitidis, Candida species (e.g. Candida albicans, Candida glabrata, Candida kerr, Candida krusei, Candida par apsilosis, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Candida stellatoidea, and Candida tropicalis), Coccidioides immitis, Conidiobolus species, Cryptococcus neoforms, Cunninghamella species, dermatophytes, Histoplasma capsulatum, Microsporum gypseum, Mucor pusillus, Paracoccidioides brasiliensis, Pseudallescheria boydii, Rhinosporidium seeberi, Pneumocystis carinii, Rhizopus species (e.g. Rhizopus arrhizus, Rhizopus oryzae and Rhizopus microsporus), Saccharomyces species, Sporothrix schenckii, zygomycetes and classes such as Zygomycetes, Ascomycetes the Basidiomycetes, Deuteromycetes and Oomycetes. In addition, fungal diseases that can be treated or prevented by the compositions of the invention include but are not limited to aspergillosis, crytococcosis, sporotrichosis, coccidioidomycosis, paracoccidioidomycosis, histoplasmosis, blastomycosis, zygomycosis, and candidiasis.

Allicin also has antiparasitic activity (Ankri S. Antimicrob. Agents Chemother. 10 (1997) pp. 2286-2288; Mirelman D. J. Infect. Dis. 156 (1987) 243-244). Thus, the compositions of the invention are useful as anti-parasitic agents, particularly for treating infections and diseases caused by parasites, for instance by protozoan parasites, including human protozoan parasites, for instance Entamoeba histolytica and Giardia lamblia. Parasitic diseases that can potentially be treated, prevented or ameliorated by the compositions of the invention include, but are not limited to, amebiasis, malaria, leishmania, coccidia, giardiasis, cryptosporidiosis, toxoplasmosis, and trypanosomiasis. Also encompassed are infections by various worms, such as but not limited to ascariasis, ancylostomiasis, trichuriasis, strongyloidiasis, toxoccariasis, trichinosis, onchocerciasis, filaria, and dirofilariasis. Also encompassed are infections by various flukes, such as but not limited to schistosomiasis, paragonimiasis, and clonorchiasis. Parasites that cause these diseases can be classified based on whether they are intracellular or extracellular. An "intracellular parasite" as used herein is a parasite whose entire life cycle is intracellular. Examples of human intracellular parasites include Leishmania spp., Plasmodium spp., Trypanosoma cruzi, Toxoplasma gondii, Babesia spp., and Trichinella spiralis. An "extracellular parasite" as used herein is a parasite whose entire life cycle is extracellular. Extracellular parasites capable of infecting humans include Entamoeba histolytica, Giardia lamblia, Enter ocytozoon bieneusi, Naegleria and Acanthamoeba as well as most helminths. Yet another class of parasites is defined as being mainly extracellular but with an obligate intracellular existence at a critical stage in their life cycles. Such parasites are referred to herein as "Obligate intracellular parasites". These parasites may exist most of their lives or only a small portion of their lives in an extracellular environment, but they all have at least one obligate intracellular stage in their life cycles. This latter category of parasites includes Trypanosoma rhodesiense and Trypanosoma gambiense, Isospora spp., Cryptosporidium spp, Eimeria spp., Neospora spp., Sarcocystis spp., and Schistosoma spp.

Allicin also has antiviral activity (Tsai Y. Planta M 5 (1985), pp. 460-461). Thus, the compositions of the invention are useful as anti-viral agents, particularly for treating viral infections and diseases. Viral infections or diseases that can potentially be treated, prevented or ameliorated by the compositions of the invention include, but are not limited to, those caused by the following viruses: human cytomegalovirus, influenza B, herpes simplex virus type 1, herpes simplex virus type 2, parainfluenza virus type 3, vaccinia virus, vesicular stomatitis virus and human rhinovirus type 2.

Owing to the enhanced antimicrobial effects of the composition of the invention, the nanoparticles comprising Allicin and the inorganic carrier can be used effectively as a supplemental component in conventional spermicidal compositions, in order to prevent or treat sexually transmitted diseases (STDs). Accordingly, the present invention further provides a spermicidal composition comprising a spermicide and nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier. Any conventional spermicide can be used in the composition.

The anticancer, antitumour and antithrombotic potential of Allicin is known (Ariga T., Biofactors 26 (2006) 93-103; and Miron T., Molecular Cancer Therapeutics 16 (2004) 275-281). Accordingly, the compositions of the invention are also useful for treating a tumour, cancer or thrombosis.

Allicin is also associated with immune system booster effects, as reported by the company Allisure™ and others (US 2007/0154581 Al, Kumar et al.). Accordingly, the compositions of the invention are also useful for stimulating or enhancing the immune system.

The composition of the invention can be used for preventing, managing, treating, or ameliorating an infection or disease (e.g. one of the above-mentioned antimicrobial infections or diseases, cancer, a tumour or thrombosis), or for stimulating or enhancing the immune system, in combination with another modality, such as a prophylactic or therapeutic agent known to be useful for, or having been or currently being used in the prevention, treatment, management, or amelioration of a disorder or used in the lessening of discomfort or pain associated with a disorder. Depending on the manner of use, the compositions of the invention can be co-administered with another modality, or the compositions or compounds of the invention can be mixed and then administered as a single composition to a subject. Thus, a composition of the invention can be added to an over-the-counter, non-prescriptional medication. Examples of such medication include but are not limited to an analgesic, acetaminophen, non-steroidal anti-inflammatory agent, salicylate, antibiotic, antidiarrheal, antihelmintic, antiemetic, antiflatulent, antifungal, antihistamine, antitussive, antimycotic, antacid, antipruritics, antipyretics, decongestant, expectorant, laxative, hemorrhoidal preparation, artificial tear, sedative, motion sickness medication, acne medication, sebborrhea medication, bum preparation, canker sore preparation, steorid, sore throat lozenge, smoke cessation aid, and wound care product. Likewise, a composition of the invention can be used in combination with a therapeutic or prophylactic agent, including, for instance, an agent selected from plant extracts, small molecules, synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g., DNA and RNA nucleotides including, but not limited to, antisense nucleotide sequences, RNAi, triple helices and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), antibodies, synthetic or natural inorganic molecules, mimetic agents, and synthetic or natural organic molecules. The therapeutic or prophylactic agent used in combination with the composition of the invention may, for example, be an anti-angiogenic agent, an anti-inflammatory agent, an anti-cancer agent, an anti-viral agent, an antibiotic, a natural product, a phytochemical or a botanical extract.

The present invention provides a pharmaceutical composition comprising nanoparticles and a pharmaceutically acceptable carrier or diluent, which nanoparticles comprise Allicin and an inorganic carrier. The composition is typically prepared following conventional methods and is administered in a pharmaceutically suitable form. The composition may be administered in any conventional form, for instance as follows:

A pharmaceutical composition of the present invention can be administered in a variety of dosage forms, for example orally such as in the form of tablets, capsules, sugar- or film-coated tablets, liquid solutions or suspensions or parenterally, for example intramuscularly, intravenously or subcutaneously. The composition may therefore be given by injection or infusion.

The dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Daily dosages can vary within wide limits and will be adjusted to the individual requirements in each particular case. Typically, however, the dosage adopted for each route of administration when a composition is administered alone to adult humans is 0.0001 to 50 mg/kg, most commonly in the range of 0.001 to 10 mg/kg, body weight, for instance 0.01 to 1 mg/kg. Such a dosage may be given, for example, from 1 to 5 times daily. For intravenous injection a suitable daily dose is from 0.0001 to 1 mg/kg body weight, preferably from 0.0001 to 0.1 mg/kg body weight. A daily dosage can be administered as a single dosage or according to a divided dose schedule.

Typically a dose to treat human patients may range from about 0.1 mg to about 1000 mg of a pharmaceutical composition of the invention, more typically from about 10 mg to about 1000 mg. A typical dose may be about 100 mg to about 300 mg of the composition. A dose may be administered once a day (QID), twice per day (BID), or more frequently, depending on the pharmacokinetic and pharmacodynamic properties, including absorption, distribution, metabolism, and excretion of the particular compound. In addition, toxicity factors may influence the dosage and administration regimen. When administered orally, the pill, capsule, or tablet may be ingested daily or less frequently for a specified period of time. The regimen may be repeated for a number of cycles of therapy. The composition may be administered in any conventional form, for instance as follows:

A) Orally, for example, as tablets, coated tablets, dragees, troches, lozenges, aqueous or oily suspensions, liquid solutions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

Tablets contain the active ingredient (the Allicin-functionalised nanoparticles) in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, dextrose, saccharose, cellulose, corn starch, potato starch, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, alginic acid, alginates or sodium starch glycolate; binding agents, for example starch, gelatin or acacia; lubricating agents, for example silica, magnesium or calcium stearate, stearic acid or talc; effervescing mixtures; dyestuffs, sweeteners, wetting agents such as lecithin, polysorbates or lauryl sulphate. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Such preparations may be manufactured in a known manner, for example by means of mixing, granulating, tableting, sugar coating or film coating processes.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is present as such, or mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials (the Allicin-functionalised nanoparticles) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinylpyrrolidone gum tragacanth and gum acacia; dispersing or wetting agents may be naturally-occurring phosphatides, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides for example polyoxyethylene sorbitan monooleate.

The said aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient (the Allicin-functionalised nanoparticles) in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.

Sweetening agents, such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by this addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.

Pharmaceutical compositions for use in accordance with the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally occuring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids an hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavouring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. In particular, a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose.

Such formulations may also contain a demulcent, a preservative and flavouring and coloring agents;

B) Parenterally, either subcutaneously, or intravenously, or intramuscularly, or intrasternally, or by infusion techniques, in the form of sterile injectable aqueous or oleaginous suspensions. This suspension may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic paternally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.

Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition fatty acids such as oleic acid find use in the preparation of injectables;

C) By inhalation, in the form of aerosols or solutions for nebulizers;

D) Rectally, in the form of suppositories prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and poly-ethylene glycols;

E) Topically, in the form of creams, ointments, jellies, collyriums, solutions or suspensions.

F) Vaginally, in the form of pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

The nanoparticles of the invention can be incorporated into materials and products in order to provide them with antimicrobial properties. Thus, for instance, the nanoparticles can be usefully employed in packaging materials and containers, where preservation of contents (e.g. by protection from microbial degradation) is important. The nanoparticles are thus usefully employed in packaging and containers for perishable foodstuffs and agricultural materials. The nanoparticles can also be used in antimicrobial surface coatings. Such coatings are particularly useful in hospitals and other medical environments, for instance, in order to reduce microbial contamination and the spread of pathogens such as MRSA. Everyday items and surfaces may be coated, such as doors, handles, floors, walls, ceilings, furniture, and work surfaces, in order to prevent or reduce microbial contamination and spread. Fabrics can be coated in addition to hard surfaces. In addition, specialist items, such as surgical instruments, surgical implants, and medical devices, may be coated with antimicrobial coatings comprising the nanoparticles. Such items could alternatively be made from materials which contain the nanoparticles of the invention, for instance polymers or plastics which contain the nanoparticles.

Accordingly, in one aspect, the invention provides a film which comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier.

The invention also provides a surface coating which comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier.

The invention further provides a coated product, which comprises a product and a coating disposed on a surface thereof, wherein the coating comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier.

The product may be any product which is desirably coated in order to reduce microbial contamination. It may for instance be a food packaging material, packaging for agricultural goods, a product for use in a hospital, a surgical implement, a surgical instrument, a medical device, or an implantable medical device such as a stent or catheter.

The film or coating in these aspects of the invention typically has a thickness of from 1 nm to 1 mm. More typically the film or coating has a thickness from 10 run to 100 μm, or, for instance, from 100 nm to 100 μm, or, for instance, from 1 μm to 100 μm.

Typically, the film or coating in these aspects of the invention comprises a bulk material and said nanoparticles. Typically, the nanoparticles are dispersed throughout the bulk material.

The bulk material typically comprises a polymer. The polymer may be a homopolymer or a copolymer, for instance a random copolymer or a block copolymer. It may thus be derived from monomeric units which are the same or different.

Typically, the polymer is selected from fluoropolymers, polyacrylates, poly(meth)acrylates, polyvinyls, polyvinyl alcohols, poly(alkylene oxides), polyesters, polyacrylics, polyamides, polyimides, polycarbonates, epoxy resins, polyethers, polyketones, polyolefins, rubbers, polystyrenics, polysulfones, polyurethanes, cellulose, polyglycoside, polypeptides, and co-polymers thereof.

The polymer may for instance be poly(vinylidenedifluoride-hexafluoropropylene) (PVDF-HFP), perfluoroalkoxy (PFA), polytetrafluoroethylene (PTFE), polyvinyl alcohol (PVA), poly(methyl methacrylate) (PMMA), polyethyleneterephthalate (PET), or poly(ethylene oxide).

In one embodiment, the polymer is a fluoropolymer. Typically, the fiuoropolymer is poly(vinylidenedifluoride-hexafluoropropylene) (PVDF-HFP), perfluoroalkoxy (PFA), or polytetrafluoroethylene (PTFE).

In one embodiment, the polymer is a ferroelectric polymer. PVDF-HFP is a ferroelectric polymer.

The polymer may be modified, for instance by admixture with another organic or inorganic material. The substrate may thus comprise both a polymer and an inorganic material, for instance a mixture of a polymer and an inorganic material such as an inorganic filler.

Additionally or alternatively, the bulk material may comprise an inorganic material including, but not limited to, a metal, a metal alloy, or a metal salt, silica, glasses, alumina, titania, a textile, paper, and allotropes of carbon such as diamond, diamond- like carbon, graphite, fullerenes and nanotubes.

The bulk material may comprise any two or more of the above-listed polymers and inorganic materials.

The product of the coated product of the invention may itself comprise a polymer, for instance any of the polymers defined above in relation to the films and coatings of the invention. Additionally or alternatively the product of the coated product of the invention may comprise an inorganic material including, but not limited to, a metal, a metal alloy, or a metal salt, silica, glasses, alumina, titania, a textile, paper, and allotropes of carbon such as diamond, diamond-like carbon, graphite, fullerenes and nanotubes.

The product of the coated product of the invention may be in any suitable physical form. Thus, the product may be in the form of a film, a layer, a sheet or a board. Alternatively, the product may be an article such as: a section or surface of a building, a sheet of glass, a piece of furniture, a surgical implement, a surgical instrument, a medical device, or an implantable medical device such as a stent or catheter. Alternatively, the product may be a textile, a powder, or in the form of, say, pellets, beads, particles, nanoparticles or microparticles. The pellets, beads or particles may be macroscopic particles, i.e. visible to the naked eye, or microscopic particles. Thus, the particles could be microparticles or nanoparticles.

In one aspect, the invention provides a packaging material, which packaging material comprises a polymer and nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier. The polymer may for instance be any of those defined above for the films and coatings of the invention. Typically, the nanoparticles are as further defined hereinbefore. Such packaging material may be used as packaging for any goods which are desirably preserved by protection from microbial degradation. The packaging material is thus usefully employed in packaging and containers for perishable foodstuffs and agricultural materials. In one embodiment, the packaging material is food packaging material. In another embodiment, the packaging material is a packaging material for an agricultural product.

Such packaging material may be produced by standard processes known in the art for producing polymeric packaging materials, the nanoparticles of the invention being introduced at any convenient stage during the process.

The invention also provides a polymer composition, which polymer composition comprises a polymer and nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier. The polymer may for instance be any of those defined above for the films and coatings of the invention. The polymer composition may further comprise one or more additional polymers, which may also, for instance, be any of those defined above for the films and coatings of the invention.

Typically, the nanoparticles are as further defined hereinbefore. The nanoparticles are typically dispersed throughout the polymer or polymers in said polymer composition. The polymers in said compositions may be produced by standard processes known in the art for producing the polymers, and the nanoparticles of the invention may be introduced at any convenient stage during the process, for instance either before, during or after polymerisation.

The invention also provides a process for producing a coating, which coating comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier, which process comprises: disposing a coating composition which comprises nanoparticles and a solvent onto a substrate, which nanoparticles comprise Allicin and an inorganic carrier; and removing said solvent. Further provided is a process for producing a film, which film comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier, which process comprises: disposing a film-forming composition which comprises nanoparticles and a solvent onto a substrate or mould, which nanoparticles comprise Allicin and an inorganic carrier; and removing said solvent. In one embodiment, the process further comprises removing the film from the substrate or mould, thereby recovering said film.

Also provided is a process for producing a coated product, which coated product comprises a product and a coating disposed on a surface thereof, wherein the coating comprises nanoparticles, which nanoparticles comprise Allicin and an inorganic carrier, which process comprises: disposing a coating composition which comprises nanoparticles and a solvent onto a product to be coated, which nanoparticles comprise Allicin and an inorganic carrier; and removing said solvent.

In the processes of the invention for producing a coating, a film or a coated product, the solvent may be any solvent in which the nanoparticles of the invention may be suitably dispersed and in which any other components present in the coating or film-forming composition may be suitably dispersed or dissolved. In one embodiment, the solvent comprises a polar protic solvent, for instance water or an alcohol. Typically, the solvent is water. In another embodiment, however, the solvent is an organic solvent.

Typically, in the processes of the invention for producing a coating, a film or a coated product, the coating composition or film-forming composition further comprises a bulk material. Typically, the bulk material is dissolved or dispersed in the solvent.

The bulk material typically comprises a polymer. The polymer may be a homopolymer or a copolymer, for instance a random copolymer or a block copolymer. The polymer may be as defined above for the films and coatings defined hereinbefore. When the bulk material comprises a polymer, the processes of the invention for producing a coating, a film or a coated product may further comprise a step of curing the polymer (but this step is by no means essential as the polymer may not require curing and/or may already be present in the composition in a fully-polymerised or fully-cured state).

Additionally or alternatively, the bulk material may comprise one or more monomers. The one or more monomers may for instance be selected from monomers of fluoropolymers, polyacrylates, poly(meth)acrylates, polyvinyls, polyvinyl alcohols, poly(alkylene oxides), polyesters, polyacrylics, polyamides, polyimides, polycarbonates, epoxy resins, polyethers, polyketones, polyolefins, rubbers, polystyrenics, polysulfones, polyurethanes, cellulose, polyglycoside and polypeptides. When the bulk material comprises one or more monomers, the processes of the invention for producing a coating, a film or a coated product typically further comprises a step of polymerising the one or more monomers to form a polymer. The resulting coating or film thus comprises a said polymer.

Typically, in the processes of the invention for producing a coating, a film or a coated product, the nanoparticles are as further defined herein.

Typically, in the processes of the invention for producing a coating, a film or a coated product, the process further comprises a preliminary step of producing said nanoparticles in accordance with the process of the invention for producing said nanoparticles, as defined herein, which comprises: treating precursor nanoparticles, comprising said inorganic carrier, with Allicin.

Typically, said bulk material is present in said composition in an amount of from 10 wt % to 80 wt % of the composition. More typically, the bulk material is present in an amount of from 40 wt % to 60 wt % of the composition.

Typically, the nanoparticles are present in the composition in an amount of from 0.1 wt % to 50 wt %. More typically, the nanoparticles are present in an amount of from 1 wt % to 20 wt %.

Typically, prior to the step of disposing the coating composition or the film-forming composition onto the substrate (or mould or product) to be coated, the process further comprises a step of homogenizing the composition. This step typically comprises subjecting the composition to shear, e.g. to agitation or stirring. Typically, the step comprises sonicating the composition.

Typically, the step of disposing the coating composition or the film-forming composition onto the substrate (or the mould or product) to be coated, comprises spraying, dip-coating or spin-coating the composition onto said substrate (or mould or product). More typically, it comprises spraying the composition onto said substrate (or mould or product). The spraying may be performed with the aid of a carrier gas. The step of spraying the composition onto the substrate (or mould or product) typically comprises (i) introducing said composition and a carrier gas into a spray head, and (ii) spraying the composition onto said substrate (or mould or product) from an exit of said spray head. The exit of the spray head typically comprises a nozzle. Thus, the spraying is typically performed through an atomizing nozzle.

Typically, the step of removing the solvent comprises evaporating the solvent. Typically, the step of removing the solvent comprises heating. Typically, the substrate (or mould or product) is heated.

The steps of disposing the coating composition or the film-forming composition onto the substrate (or mould or product) and heating the substrate (or mould or product) are typically performed simultaneously.

Typically, the steps of disposing the coating composition or the film-forming composition onto the substrate (or mould or product), and removing the solvent are performed or repeated until a film or coating having a desired thickness is formed. Typically, the thickness of the film or coating is from 10 nm to 100 μm, or, for instance, from 100 nm to 100 μm, or from 1 μm to 100 μm.

The present invention is further illustrated in the Examples which follow:

EXAMPLES

Example 1 - Production of Allicin-Gold antimicrobial nanomaterial

Fresh Garlic from a local Oxford store was crushed to activate the Allinase enzyme contained in the garlic cloves to react with Alliin (chemical found in the garlic cloves) to form Allicin. A schematic illustration of this reaction is shown in Fig. 1. Once the garlic cloves were crushed, the Allicin was extracted into an aqueous buffer solution containing 20% ethanol, to form a suspension containing Allicin. The suspension was filtered with a 0.45 microns pore size filter, and the filtrate suspension was subjected to a centrifugation purification step at 4000 revolutions per minute (RPMs) for 5 minutes. An extra purification step using an HPLC is recommended for quality control of the commercial product. The resulting Allicin solution, in water with 20% ethanol, was stored at 4 ⁰ C, ready for the functionalization steps.

Chloroauric acid, H[AuCl ₄ ], and Na-citrate were purchased from Sigma- Aldrich UK at their highest purities respectively. Chloroauric acid was dissolved in purified water at a concentration of 0.1w/v % (e.g. 0.1 g in 100 mL of water). The resulting chloroauric acid solution was heated-up to boiling, with vigorous stirring. 20 v/v % of the extracted and purified Allicin solution was then added to the chloroauric acid solution. This was followed by the addition of sodium citrate at a concentration of 0.5 % w/v, to enhance the reduction of gold and functionalize the Allicin. Allicin was added first, i.e. before the addition of sodium citrate, to ensure uniform functionalization of Allicin. After the addition of Allicin, the solution underwent a rapid colour change from light yellow to a purple/red wine color, including the formation of colloidal gold, after which the sodium citrate was added. After the addition of sodium citrate, the solution was kept under agitation and heated for another 45 minutes. The resulting Allicin-functionalised nanomaterial solution (a dispersion of Allicin-functionalised colloidal gold nanoparticles) was then cooled down to room temperature, filtered using a 0.45 microns filtration system, and then centrifuged for 10 minutes at 3000 RPMs. The resulting filtered and centrifuged liquid, containing the Allicin-gold nanoparticles, was stored at 4 ⁰ C.

Fig. 2 shows a schematic illustration of the reduction of [AuCl ₄ ] ^" to form gold nanoparticles in the presence of Allicin, to produce the Allicin-functionalised gold nanoparticles of the invention.

Example 2 - Production of Allicin-Zeolite nanomaterial

Nanocrystalline zeolites with a chemical formula of [K ⁺ ₆ Na ⁺ ₃ (H ₂ O) _2I ] [Al ₉ Si ₂₇ O ₇₂ ] (purchased from Nanoscape, Germany) were suspended in water at a concentration of 10% w/w to form a pure aqueous suspension. 20% v/v of a purified Allicin solution, made from crushed garlic using a similar procedure as outlined above in Example 1 , was added to the suspension of Zeolite nanoparticles. 1 % v/v of aqueous hydrochloric acid (HCl, Sigma, UK) was then added to the suspension to stimulate ion exchange between the suspended zeolites and Allicin. The suspension was homogenized in a mixer at 60 RPMs for 4 hours at room temperature to ensure good ion exchange in the system. The resulting final suspension, comprising Allicin-functionalised zeolite nanoparticles, was stored at 4° C.

Fig. 3 shows a schematic illustration of the ion-exchange reaction between Allicin and zeolite.

Example 3 - Characterisation of Allicin-gold and Allicin-zeolite nanoparticles

(a) Zeta potential The zeta potentials of the Allicin-gold and Allicin zeolite nanoparticles were measured, and compared to those of the respective starting materials, i.e. to those of nano gold, zeolite nanoparticles and Allicin alone.

The zeta potential of the Allicin-functionalized nano-gold was in the range of -1 OmV to -8mV. The zeta potential of the Zeolite- Allicin nanoparticles was approximately -20 mV. These zeta potential values reflect the slightly negative anionic charge on the nanoparticle surfaces as well as the high stability of the systems. This zeta potential stability contributes to the increased stability of the antibiotic nanoparticle compositions of the invention over long periods of time, ensuring that the quality (activity) of the antibacterial compound does not diminish over time.

Figures 4 and 5 show the zeta potentials of the functionalized Allicin-gold and Allicin- zeolite nanoparticles respectively. Figures 6, 7 and 8 show the zeta potential distributions for Allicin, zeolite nanoparticles and gold nanoparticles respectively. The zeta potential measurements show the effects of Allicin on the charge of the system when interacting with nano-gold and zeolite suspensions respectively. The high anionic strengths of the zeta potentials of gold nanoparticles and zeolites resectively complement the low zeta potential of Allicin, making these systems more stable over long periods of time. From Figures 4 to 8 it can be ascertained that the zeta potentials are as follows: Allicin alone = -6.66 mV; gold nanoparticles = -38.0 mV; zeolite nanoparticles = -54.5 mV; nanogold-Allicin compound = -8.68 mV; and zeolite-Allicin compound = - 21.2.mV.

(b) X-ray Photoelectron Spectroscopy (XPS)

XPS was performed to analyze the chemical composition of the nanomaterial as a way to verify the presence of Allicin and the inorganic carrier (e.g. Au). Figure 9 shows the X-ray photoelectron spectrum of a composition comprising Allicin- functionalised gold nanoparticles in accordance with the invention. XPS confirmed the presence of Allicin (due to the sulphur peaks) as well as gold.

(c) Imaging of gold nanoparticles functionalized with Allicin Figures 10, 11 and 12 were taken in a JEOL 2010 20OkV analytical electron microscope (AEM). They show the gold spherical nanoparticles functionalized with Allicin, after the functionalisation procedure via the modified reduction method of the present invention. Figure 10 shows gold spherical nanoparticles functionalized with Allicin, in accordance with the invention. The average size (diameter) of the gold nanoparticles (the dark regions), is under 20nm, which is thought to enhance the antibactericidal effect of Allicin by increasing the surface area of contact with Allicin. Figures 11 and 12 show Allicin- functionalised gold nanoparticles; it is clear from these Figures that the particles do not tend to agglomerate, which is consistent with the zeta particle measurements of these particles, the relatively high magnitude of the zeta potentials also indicating the the particles do not coagulate or floculate but are electrically stable. Furthermore, the uniform shape and size of the nanoparticles demonstrates the suitability of the particles for effective attachment to natural compounds.

Example 4 - Antibiotic Disk Diffusion Tests

Disc diffusion is a method of antimicrobial susceptibility testing. In this test, small sterile filter paper disks (Fischer, UK) with a diameter of 6 mm were placed on an agar nutrient plate inoculated with a lawn of bacteria of different bacterial strains including Gram negative E. coli and Gram positive B. subtilis. The disks were impregnated with 100 μL of the Au-Allicin and Zeolite-Allicin compounds respectively and then they were incubated at 37°C for 48 h.

The zone of inhibition of bacterial growth (diameter of inhibition) is used as a measure of susceptibility. In general, a larger area of inhibition indicates that the organism is susceptible, whilst a smaller area or "no zone" of inhibition indicates resistance to the antibiotic material.

The potential of nanomaterials functionalized with Allicin as antibacterial agents is demonstrated in this Example, in which a 50-fold improvement in the minimum inhibition concentration is observed. At concentrations as low as 4 μg/ml the E. coli strain showed results comparable to Allicin at concentrations of 200-300 μg/ml (see in particular Figures 13 and 14). The Au-Allicin and zeolite- Allicin extracts were kept in a refrigerator at 4°C for a period of a month to test the antibacterial properties of these antibacterial nanomaterials over time. Even though Allicin usually loses all its antibacterial activity in less than a month, our new stabilized and protected nanomaterial-Allicin compounds show remarkable antibiotic potential. This effect can be see in Figures 15 and 16, which relate to Gram negative and Gram positive bacteria respectively.

Antibacterial effect on further Gram positive and Gram negative bacteria

Compositions of the invention comprising Allicin-functionalised nanoparticles were tested on the following further strains of bacteria, showing a remarkably low MIC in each case:

Staphylococcus sp. (Gram positive) Arthrobacter sp. (Gram positive) Flavobacterium sp. (Gram negative) Rhodococcus sp. (Gram positive) Pseudomonas sp. (Gram negative) E. coli sp. (Gram negative) Bacillus sp. (Gram positive)

Antifungal effect

Compositions of the invention comprising Allicin-functionalised nanoparticles were tested on the following strains of fungi, exhibiting remarkably low MIC values: Aspergillus sp., including Aspergillus niger and Aspergillus terreus. Another important fungal species susceptible to Allicin includes Candida albicans.

Resuspension experiment

The antimicrobial gold and zeolite nanomaterials functionalized with Allicin were dried to their powder forms and then resuspended in ultra pure water (filtered with a 0.2 micron filter). The Allicin concentrations per milliliter were quantified using the modified Allicin assay (see below) prior to disk susceptibility testing on Gram-positive and Gram-negative bacteria as well as in fungi strains.

The following bacterial strains were tested to measure the antibacterial potential after rehydration of the powder, both for the nanogold-AUicin nanomaterial (Allicin concentration, [2 μg/mL]) and zeolite- Allicin nanomaterial (Allicin concentration, [0.5 μg/mL]):

Controls

Two control compositions, containing unfunctionalised gold nanoparticles and zeolite nanoparticles respectively, were subjected to antibiotic disk diffusion texts. Both control compositions showed no antimicrobial effect on all the bacterial and fungi species tested.

Allicin concentration and Stability

Allicin concentration in the functionalized gold or zeolite drug carriers was measured following a modified spectrophotometric assay for Allicin with the chromogenic thiol 4- Mercaptopyridine (Miron, 2002). The chromogenic thiol 4-Mercaptopyridine (4-MP) reacts selectively under the presence of Allicin and if Allicin is present, the absorbance spectrum of 4-MP shifts upon reacting with it (4-Mercaptopyridine, λ=324nm). The modified assay was tested in the different functionalized antimicrobial solutions for several weeks. Data shows that the protective value of gold and zeolite stabilizes the solution and retains the antimicrobial activity of Allicin. In fact, the conjugated effect of Allicin protected either by a gold carrier or a zeolite allows the antimicrobial compound to perform with remarkable antibiotic potential.

Example 5 - Kinetic studies

The kinetic studies whose results are summerised in Figures 17 to 20 were performed in a BioteK Synergy™ HT Multi-Mode Microplate Reader for a duration of 24 h for different bacterial strains. Bacteria were inoculated in nutrient broth and incubated at a temperature of 37°C. The microplates used for these experiments were sterile BD Falcon™ 96-well Microplates. The growth of the bacterial strains over time was measurd by measuring the absorption units at λ = 600 nm.

The graphs of Figures 17 to 20 have been normalized and all the experiments were performed in triplicate, for statistical accuracy and reliability. Figure 17 shows the antibiotic effect of a composition of the invention comprising gold/Allicin nanoparticles on Gram negative bacteria E. coli. (Note: The average standard deviation of the Fig. 17 plot was: E. coli control = 0.015, Au:AUicin = 0.01). Figure 18 shows the antibiotic effect of a gold/Allicin nanoparticle composition of the invention on Gram positive bacteria B. subtilis. (Note: The average standard deviation of the Fig. 18 plot was: B. subtilis control = 0.014, Au:Allicin = 0.02). The results for the Au-Allicin nanoparticle composition are similar for both Gram positive and Gram negative bacteria. The Au-Allicin composition is several-fold more stable and effective than Allicin alone, underlining the bactericidal potential of the compositions of the invention. Figure 19 shows the antibiotic effect of a composition of the invention comprising Zeolite- Allicin nanoparticles on Gram negative bacteria E. coli. (Note: The average standard deviation of the Fig. 19 plot was: E. coli control = 0.015, Au:Allicin = 0.006). The results of the functionalisation of Allicin with Zeolites show a powerful bactericidal effect. Figure 20 shows the antibiotic effect of a composition of the invention comprising Zeolite- Allicin nanoparticles on Gram positive bacteria B. subtilis. (Note: The average standard deviation of the Fig. 20 plot was: E. coli control = 0.015, Au: Allicin = 0.006). Again, a potent bacetericidal effect was shown. Example 6 - Case Study: E. CoIi

An E. coli strain was treated with the Au- Allicin and (separately) the Zeolite-Allicin antibacterial composition of the invention. After 60 minutes, each composition demonstrated a considerable bacterial killing rate (see the viability test section below). TEM images (Figs. 21 and 22) show the differences between a control untreated E. coli strain and a strain treated with the Au- Allicin or Zeolite-Allicin compound. Note the well- defined cell wall membrane and overall healthy appearance of the cells prior to interaction with the antibacterial nanomaterial compound (Figure 21 (a) and (b)). The changes in the permeability of the cell wall are evident from Figures 22 (a) to (d). These Figures show that the Au- Allicin and the Zeolite-Allicin compounds have aggressively activated the ion channels of the cell membrane, which increases the permeability of the bacterial cell wall. As a result of this increase in the permeability of the cell wall some cells have a bloated appearance with evident stresses in the cell wall. The TEM pictures were taken in an 8OkV Phillips Tecnai Tl 2 FEI electron microscope and show the devastating effects of the antimicrobial nanomaterials (Au- Allicin, Zeolite-Allicin) developed. The most likely mode of action for the antibiotic compounds involves the dynamic activation of ion channels of the cell, to dramatically increase permeability across the cell membrane, thereby deactivating and killing the target cell.

Cell viability

An E. coli strain was treated with nanogold- Allicin and Zeolite-Allicin antibacterial compounds at the logarithmic stage of growth. An Invitrogen Live-Dead iføcLight bacterial viability kit was used to detect the percentage of live bacteria immediately after exposure to the nanogold-Allicin or Zeolite-Allicin compound.

Figures 23 and 24 demonstrate the antibacterial potency of these functionalized compounds at very low concentrations of Allicin. Similar behavior was seen in other Gram positive and Gram negative bacteria. Example 7 - Tablet composition

Tablets, each weighing 0.15 g and containing 25 mg of nanoparticles comprising Allicin and the inorganic carrier, are manufactured as follows:

Composition for 10.000 tablets

Nanoparticles comprising Allicin and the inorganic carrier (250 g) Lactose (800 g) Corn starch (415 g) Talc powder (30 g) Magnesium stearate (5 g)

The nanoparticles, lactose and half of the corn starch are mixed. The mixture is then forced through a sieve 0.5 mm mesh size. Corn starch (10 g) is suspended in warm water (90 ml). The resulting paste is used to granulate the powder. The granulate is dried and broken up into small fragments on a sieve of 1.4 mm mesh size. The remaining quantity of starch, talc and magnesium is added, carefully mixed and processed into tablets.

Example 8 - Injectable Formulation

Formulation A

Nanoparticles comprising Allicin and inorganic carrier 200 mg

Hydrochloric Acid Solution 0.1M or

Sodium Hydroxide Solution 0. IM q.s. to pH 4.0 to 7.0

Sterile water q.s. to 10 ml

The nanoparticles are dispersed in most of the water (35° to 40° C) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch is then made up to volume with water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals. Formulation B

Nanoparticles comprising Allicin and the inorganic carrier 125 mg Sterile, Pyrogen-free, pH 7 Phosphate

Buffer, q.s. to 25 ml

Active compound 200 mg

Benzyl Alcohol 0.10 g

Glycofurol 75 1.45 g

Water for injection q.s to 3.00 ml

The nanoparticles are dispersed in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).

Example 9 - Syrup Formulation

Nanoparticles comprising Allicin and the inorganic carrier 250 mg

Sorbitol Solution 1.50 g

Glycerol 2.00 g

Sodium benzoate 0.005 g

Flavour 0.0125 ml

Purified Water q.s. to 5.00 ml

The nanoparticles are dispersed in a mixture of the glycerol and most of the purified water. An aqueous solution of the sodium benzoate is then added to the solution, followed by addition of the sorbitol solution and finally the flavour. The volume is made up with purified water and mixed well.

Example 10 - Production of antimicrobial polymer films comprising allicin-loaded nanoparticles

Summary

Antimicrobial testing was done using two common fluoropolymers, (poly (vinylidenedifluoride-hexafluoropropylene) copolymer (PVDF-HFP) and Perfluoroalkoxy (PFA) showing effective antimicrobial inhibition on polymeric films loaded with Allicin loaded nanoparticles.

Polymers tested

The PVDF-HFP is a ferroelectric polymer with high dielectric constant and widely used in microelectronic materials. PFA is a common fluoropolymer similar in composition and properties to the fluoropolymer polytetrafluoroethylene (PTFE) also known by its brand name Teflon

Methods

An approximately 50 wt% solid aqueous dispersion of the fluoropolymer of choice was mixed with Allicin-loaded nanoparticles (Allicin concentration 2 μg/mL), achieving a homogeneous solution containing 10 wt % of Au- Allicin nanoparticles and they were vigorously mixed and sonicated to reduce agglomeration in an ice bath using a probe and bath ultrasonication unit. After mixing, the solutions were sprayed onto a preheated glass substrate using a spray coating unit. The solutions were sprayed with a nozzle via a hypodermic needle by a syringe pump and the solutions were atomized into droplets by an atomizing nozzle operating with compressed air at 140 kPa. The glass substrate sat on a heated table that moved in the x-y plane (speed of 100 mm/s). The atomized droplets produced a fine mist on the glass substrate which was then cured into a film in a belt furnace. This spray-drying-curing process was repeated as required to build-up film thicknesses of approximated 30 μm for the antimicrobial tests. The resulting films were cut in small squares and place on bacteria inoculated (gram positive and gram negative bacteria) agar plates using standard antimicrobial testing procedures and in aseptic conditions.

Results

The results of the antimicrobial testing showed bacterial inhibition in all the bacterial strains tested including gram positive bacteria Bacillus subtilis and Staphylococcus aureus. The same effects were seen in gram negative bacteria E. coli and Pseudomonas aeruginosa. Figure 25 shows the results against the two representative gram negative and gram positive bacterial strains E. coli and B. subtilis respectively.