Powder Formulations For Controlled Release Of Reactive Oxygen Species

Abstract

The invention discloses a metal and semi-metal oxide powder that, when applied to an environment, inhibits the growth of colonies of microorganisms, wherein the powder includes particles comprising a particle size distribution between 0.1 to 100 microns, which are formulated as a strongly bonded, porous, composite of nano-scale grains of materials wherein the grains have a surface area of 75 to 300 m.sup.2/g and which have less than about 10.sup.−4% of free radical species by weight, and wherein the powder is adapted to release reactive oxygen species (ROS) burst when the particles come into contact with a microorganism.

Claims

1. A metal and semi-metal oxide powder that, when applied to an environment, inhibits the growth of colonies of microorganisms, wherein the powder includes particles comprising a particle size distribution between 0.1 to 100 microns, which are formulated as a strongly bonded, porous, composite of nano-scale grains of materials wherein the grains have a surface area of 75 to 300 m.sup.2/g and which have less than about 10.sup.−4% of free radical species by weight, and wherein the powder is adapted to release reactive oxygen species (ROS) burst when the particles come into contact with a microorganism.

2. The powder of claim 1, wherein the particle size distribution is between 1 to 20 microns.

3. The powder of claim 1 or claim 2, wherein the microorganisms include a biofilm and wherein the acidity of the biofilm triggers the release of the ROS burst which then suppresses the growth of the microorganisms.

4. The powder of claim 1 or claim 2, the powder is adapted to be used in one of the following environments: a marine environment, a sewer crown environment, a plant, an animal, or a human.

5. The powder of claim 1 or claim 2, wherein the microorganisms are selected from one of the following group: viruses, bacteria, fungi or larvae of insects.

6. The powder of claim 4, the metal oxide is selected from one of the following oxides: AgO, ZnO, CuO, MgO, SiO.sub.2, Al.sub.2O.sub.3, Mn.sub.3O.sub.4, and wherein the respective positive ion is selected to provide nutrients to the selected environment.

7. The powder of claim 6, wherein the powder includes MgO and the powder inhibits the microorganism growth by the suppression of hydrogen sulphide, ammonia and phosphorous produced by the microorganisms.

8. The powder of claim 6, the powder includes less than 1% of the maximum amount of radical species by weight and wherein the powder is generated by annealing the unprocessed powder at a calcination temperature within the range of 400 to 800° C.

9. The powder of claim 6, the powder includes less than 1% of the maximum amount of radical species by weight and wherein the powder is generated by hydration of the unprocessed powder in 0.01M citric acid.

10. The powder of claim 6, wherein the powder includes the following characteristics: a. A porosity of the particles is in the range of 0.3 to 0.5; and b. A specific surface area is in the range of 75 to 300 m.sup.2/g; and c. A mean grain size of the powder is in the range of 5-20 nm; and d. A strength characterised by a high resistance to grinding attributed to the binding of grains in the composite by necks that are less than about 1 nm in size, and a Youngs modulus of 5% of that of the equivalent bulk material.

11. The powder of claim 9, wherein the powder is produced by calcination at a temperature within the range of 400 to 800° C.

12. The powder of claim 10, wherein the powder is adapted for use in a marine coating which inhibits microorganism growth on the coating.

13. The powder of claim 10, wherein the powder is a component of a coating applied to sewer crowns which inhibits the growth of Sulphur Oxidising Bacteria.

14. An oxide powder comprising micron-sized calcined particles, wherein nano-active properties are induced in the particles during the calcination process, and wherein reactive oxygen species (ROS) present on the surface of the nano-active particles are generated in a burst mode triggered when the calcined particles contact a pathogenic microorganism.

15. The oxide powder of claim 14 wherein the calcined particles comprise strained crystals formed during the calcination process to store energy in the crystal structure.

16. The oxide powder of claim 15 wherein the energy stored in the crystal structure of the particles is released to form the burst of reactive oxygen species when the particles are contacted by H.sub.3O.sup.+ acid species from an active biofilm associated with the pathogenic microorganism.

17. The oxide powder of any one of claims 14 to 16 wherein the oxide is selected from the group comprising: AgO, ZnO, CuO, MgO, SiO.sub.2, Al.sub.2O.sub.3, Mn.sub.3O.sub.4, or mixtures thereof.

18. The oxide powder of any one of claims 14 to 17 wherein the reactive oxygen species is selected from the group comprising: hydrogen peroxide, superoxide, or peroxy radicals.

19. The oxide powder of any one of claims 14 to 18 wherein the calcined particles have an approximate average diameter of between 1 to 10 microns.

20. The oxide powder of any one of claims 14 to 19 wherein the powder is produced by calcination at a temperature within the range of 400 to 800° C. for a time period of less than 30 seconds and then quenched after calcination.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0053] Embodiments of the invention will be better understood and readily apparent to one of ordinary skill in the art from the following written description, by way of example only, and in conjunction with the drawings, in which:

[0054] FIG. 1 illustrates a schematic of an embodiment that illustrates a nano-active particle interacting with the biofilm exuded by a microcolony of pathogenic microorganisms. In FIG. 1A the illustration shows the initial contact of the particle with the biofilm and its active bacteria, and FIG. 1B shows the decomposition of the particle through the reaction of the acidic water in the biofilm, and the release of ROS into the film and its diffusion into the active cells of the biofilm, and FIG. 1C shows the partially decomposed particle in the presence of the deactivated cells.

DESCRIPTION OF THE INVENTION

[0055] Preferred embodiments of the invention will now be described by reference to the accompanying drawings and non-limiting examples. It is emphasised that the mechanism described by the embodiment of FIG. 1 for the case of bacteria biofilms is similar to embodiments for colonies of virus, anaerobic fungi and the larvae of insect pests.

[0056] In a first preferred embodiment of the present invention, an example used to describe the invention is the interaction of a nano-active particle 101 with a biofilm. Biofilms have a characteristic structure consisting of bacterial microcolonies enclosed in a hydrated matrix of microbially produced proteins, nucleic acids, and polysaccharides. The bacterial cells in a biofilm are significantly more resistant to environmental stresses or microbially deleterious substances, such as antibiotics, and biocides, than planktonic cells. The development of a biofilm involves the reversible attachment of bacterial cells to a surface, followed by the irreversible attachment mediated by the formation of exopolymeric material, then formation of microcolonies and the beginning of biofilm maturation. During maturation, a 3-dimensional structure containing cells packed in clusters with channels between the clusters that allow transport of water and nutrients and waste removal, and lastly, cells detach and disperse to initiate new biofilm formation. FIG. 1 describes the case in which the nano-active particle 101, preferably produced by flash calcination and quenching described below, interacts with the growing exopolymer matrix 102 exuded from the bacterial cells 103-106.

[0057] The nano-active particle may be a metal oxide material, such as MgO, and is generally and preferably 1-10 microns in size and is similar in size to the bacterial cells. The material is preferably made using the flash calcination and flash quenching process described by Sceats et. al. in which a precursor material, comprising volatile constituents of about 30-60% by weight, is flash heated in a reactor to a temperature in which the decomposition occurs as quickly, and at as low a temperature, as possible to produce the porous metal oxide, and then rapidly quenched to prevent sintering. In this embodiment, the reaction conditions are optimised such that the grains of the metal oxide are comprised of crystals that are highly strained. Such a strain can be observed by the displacement of characteristic X-ray diffraction peaks from those of an annealed material of the same chemical composition. The diffraction lines are broadened by the small number of crystal cells in the grains, and also by the strain gradients in the grain cells. The stress energy is stored in the particle and is later used to create the ROS species. An important consequence is that the energy from the lattice strain in the particle is not released during the production process by the formation of oxygen vacancies, but rather, the energy is stored in the particles are released to form ROS species when the particle is attacked by the acid species from an active biofilm.

[0058] The condition for this process is the minimisation of the temperature of the calcination process and the minimisation of the residence time at that temperature, so that the displacement of an oxygen atom within the grain is not activated during the production process. The achievement of that criterion is the measurement of paramagnetic species using electron paramagnetic resonance (EPR) spectroscopy, because the ejection of an oxygen atom to form an oxygen vacancy from within the grain generally leaves behind an electron at the vacancy site, as a paramagnetic F-centre, and the ejected oxygen ion O.sup.− is bound to the surface of the grain as a paramagnetic V-centre. The criterion is achieved if the characteristic EPR signal of these species is not observed. For the avoidance of doubt, the product may be heated and the characteristic EPR spectrum is observed during the subsequent sintering of the particle. That sintering is also characterised by reduction of the surface area of the grains, as measured by the specific surface area of the material (in m.sup.2/g), and a narrowing of the X-Ray diffraction lines as the strain is relieved and the grains grow. In summary, the flash calcination/flash quenching process technology is operated to minimise the release of stress and the minimum production of paramagnetic species in the nano-active material particles. Such operating conditions are generally different for different precursors materials because the energetics for decomposition of the precursor and activation of the generation of oxygen vacancies are different. The temperature for calcination is preferably 50° C., or most preferably 10° C. above the equilibrium temperature of the decomposition reaction of the precursor at pressures of the volatile gas, that are as low as practically possible for an industrial process.

[0059] The biofilm 102 is generally acidic in nature, where the low pH arises from the conversion of sugars by the bacteria into energy to form the biofilm. This pH may be in the range of 4.5-6.5 in an active biofilm, compared to about 7.0 for a quiescent biofilm. The metal oxide has a pKb that depends on the chemical composition. For example, that of MgO is about 10.4. It has a low solubility in neutral water, and a slow dissolution rate at ambient temperature. However, the rate of reaction accelerates when the pH is acidic, and as a consequence, MgO powder neutralises the acid initially by the formation of Mg(OH).sub.2. The invention described herein is associated with the observation that the attack of the powder by the acid causes the formation of the radical F and V centre species, as observed by the growth of the EPR spectrum characteristic of such species as the particle reacts. Without being bound by theory, it is apparent that the reaction with H+ induces a stress relaxation of the grains of the metal oxide as the crystal structure changes from the oxide form to the hydroxide form. This is not unexpected because the crystal structures of the oxide is generally different from that of the hydroxide so that transport of oxygen is required to enable the phase change. The high strain of the oxide crystal promotes the rate of the lattice reconstruction.

[0060] It is well established that the reaction of F and V centres with water leads to the production of ROS. Indeed, the EPR technique can be used to quantify the concentration of ROS species in the particle, by use of spin traps such as DPPO in H.sub.2O.sub.2 which forms stable radical species with a characteristic EPR spectrum when ROS is present. Most significantly, the concentration of ROS observed in the reaction of an oxide powder is higher than that observed in the same material which has been completely hydrated, for example by fast hydration at a higher temperature. Without being limited by theory, the hydroxide material is a carrier of ROS which is stored in the hydroxide powder, whereas the oxide material generates ROS when it is hydrated in situ under attack by the acid from the biofilm, and in this case, the release of ROS is quantitatively higher than the stored ROS because the radical recombination which generally occurs to lower the ROS competes with the outward diffusion of the ROS to attack the structures in the bacterial cells, which turns off the generation of acid. In effect, the acid from the active bacterial cells making biofilm triggers the release of ROS which turns off the generation of biofilm. This is termed an ROS burst because it mimics the ROS burst response of a eukaryotic cell to the presence of biofilm exuded by pathogens. The concentration of ROS in the particle measured by EPR spin trap measurements is preferably the order of micromolar, which is the same order of magnitude as found in the burst of ROS from eukaryotic cells. This is an important factor because higher concentrations of ROS would attack the structures in such cells. This can be controlled by the process conditions and the application rate to reduce infection so that the ROS burst has sufficient intensity to turn off the bacterial cells, but insufficient intensity to damage the eukaryotic cells of animals and plants. Under those conditions, the material is non-toxic to animals and plants, while inhibiting disease.

[0061] It is noted that H.sub.2O.sub.2 is a volatile constituent of ROS, and the presence of H.sub.2O.sub.2 in the air near a biofilm or a partly hydrated surface of a nano-active particle may inhibit insect pests from attack. Further, any suppression of H.sub.2S or NH.sub.3 from a biofilm inhibited by the nano-active material may also be signalling factor for inhibition of pests.

[0062] Thus, turning to FIG. 1 as an example embodiment. This a schematic of the evolution of a system in which a nano-active, highly strained, particle 101, typically 1-20 microns dimeter in an aqueous medium 102 becomes initially engaged (A) with a biofilm 103 around bacteria 104-107. The bacteria are active, for example creating additional biofilm, and exude acid, represented as H.sub.3O.sup.+ into the biofilm from such synthesis processes as shown in (B) and there is a flux of acid that moves towards the alkali particle which hydrates and neutralises the acid, as show schematically by a thin layer 108 which eats into the particle. The transformation of the particle from an aggregate of metal oxide crystalline grains to an aggregate of hydrated crystalline or amorphous grains required atomic rearrangement and in this case, the oxygen atoms are ejected from the oxide to form radical species, such as the F and V centres previously described. These radical species react with water in the layer 108 to generate ROS species such as the hydroxyl radical, .OH, hydrogen peroxide H.sub.2O.sub.2, and superoxide ions .O.sub.2.sup.− and these diffuse into the aqueous layer and the biofilm, and a flux of ROS moves towards and into the bacteria where they react, and begin to switch off the power generation mechanism of the bacteria. As a consequence, the system evolves to (D) where the bacteria 104X-107X have become quiescent (or killed), and the absence of the acid in the biofilm slows down the hydration reaction and the ROS generation, as shown in D so that the consumption of the nano-active particle 101 slows down after having consumed an amount of the particle 108X in reacting with the acid and producing the ROS. The result of these reactions consumes the particle (not shown) through the release of Mg.sup.2+ ions into the solution to balance the charge from neutralisation and RO generation. In reality, the reactions generally occur throughout the porous particle. The end effect is that the nano-active powder has quenched the formation of biofilm in this example embodiment, so that disease is inhibited. It is stressed that the inhibition does not necessarily have to kill the bacteria as would a true toxic chemical bactericide, so that the bacteria and the eukaryotic cells (if present) can coexist.

[0063] It would be appreciated by a person skilled in the art that the interaction between the cells of animals and plants with various diseases from microorganisms and insects is very complex, and has evolved over time with a wide variety of responses including biofilms and extracellular matrices, including the involvement of ROS as described above. Thus, the example embodiment of FIG. 1 is one example of how a nano-active particle may interact with a pathogen, including viruses, bacteria and fungi. It is appreciated by people skilled in the art that there is a hierarchy of complexity in the structures of such microorganisms that lead to a more complex picture than that described in FIG. 1.

[0064] The general approach described in the particular embodiment applies to a wide variety of metal and semi-metal oxides, including AgO, ZnO, CuO, MgO, SiO.sub.2, Al.sub.2O.sub.3, Mn.sub.3O.sub.4 and others. The particular precursors may be carbonates, hydroxides, amines, and hydrated oxides. The nano-active materials may be produced as strained oxides in a variety of ways from precursors, including flash calcination and quenching, and the paramagnetic defect centres may have different hydration rates, pH equilibria, and activation energies for formation of radical species by mechanisms similar to oxygen atom displacement described above.

[0065] In the case of MgO the strained nano-active MgO particles may be formed from magnesium carbonate by flash calcination and quenching, and the ROS is generated during the hydration reaction. It is been established that the residual MgO, or Mg(OH).sub.2 may play other roles in the process. For example, as an alkali, the particles change the local pH in such a way to minimise the formation of H.sub.2S, a known toxin, from the decomposition of materials, such a proteins. Furthermore, the porous MgO is known to extract phosphorous through the formation of magnesium phosphates, and phosphorous and ammonia from the formation of struvite, MgNH.sub.4PO.sub.4.6H.sub.2O. In the context of biofilms, these processes may occur after the ROS production from hydration of the MgO, and may promote a favourable ecosystem for the system. It is generally accepted that anaerobic and aerobic organisms and microorganisms survive together in such systems in a symbiotic relationship. Thus the linkage between particle size distribution, ROS generation, degree of hydration, solubility and particle consumption, alkalinity, H.sub.2S inhibition, phosphate and struvite production are complex. In this context, the particles from an initially porous high surface area MgO accelerate reactions, and can act as nuclei to accelerate the formation of materials, such a struvite, that otherwise require seeding with struvite nuclei to induce precipitation, or precipitation occurs under abnormal conditions such as pressure gradients at bends which form struvite films in bends of pipes in reactors such as digesters.

[0066] Notwithstanding these complexities, it is observed that there is a substantial increase in efficacy of control on pathogens between formulations particles that had been minimally hydrated prior to inoculation, compared to particles that had been fully hydrated before inoculation, where all other variables are substantially the same. That impact in this invention, is attributed to the ability of a strained metal oxide particle to release a burst of ROS in the presence of active microorganisms at a higher ROS concentration that particles with stored ROS formed by prior hydration.

[0067] Further forms of the invention will be apparent from the description and drawings.

[0068] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.

[0069] The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable.