Metal oxides nanoparticles and uses thereof

Abstract

The present disclosure provides a metal oxide nanoparticle comprising a metal oxide, the metal capable of being a Lewis acid; a polymer comprising an ionisable moiety; and an amino acid; wherein a weight ratio of amino acid to 5 polymer is about 0.1:99.9 to about 99.9:0.1; wherein the amino acid is electrostatically interacting with the polymer; wherein the metal oxide comprises metal ions in a 3+ and 4+ valence state; and wherein the metal oxide is selected from lanthanide oxide and / or zirconium oxide. The present disclosure also provides a method of fabricating a metal oxide particle and use thereof.

Description

[0001]

[0002] Metal Oxides Nanoparticles and Uses Thereof

[0003] Technical Field

[0004] The present invention relates, in general terms, to metal oxides nanoparticles and their uses thereof.

[0005] Background

[0006] During inflammatory diseases and / or wounds, the release of elevated extracellular ATP (eATP) from immune cells and damaged / dead cells serves as a potent 'danger' signal. This eATP activates the P2X7 receptor on immune cells, intensifying the inflammatory response by enhancing phagocytosis, inducing inflammasome formation, and promoting proinflammatory cytokine release, thereby exacerbating inflammation. Although small-molecule antagonists targeting P2X7 have demonstrated therapeutic benefits in various preclinical models of inflammatory diseases, nevertheless, their lack of specificity and undesirable side effects present challenges. Additionally, solely blocking ATP receptors may not be optimal, as extracellular ATP removal may not be immediate and effective in all patients, particularly those with decreased ATP- hydrolyzing ectoenzyme expression.

[0007] Metal Organic Framework (MOF) type nanomaterials have been used for ATP clearance. However, such nanomaterials have large sizes, poor solution dispersibility, low activity and stability, and exhibit high toxicity when applied in cell experiments.

[0008] It would be desirable to overcome or ameliorate at least one of the abovedescribed problems.

[0009] Summary

[0010] The present disclosure provides a metal oxide nanoparticle comprising: a) a metal oxide, the metal capable of being a Lewis acid; b) a polymer comprising an ionisable moiety; and c) an amino acid; wherein a weight ratio of amino acid to polymer is about 0.1 :99.9 to about 99.9:0.1; wherein the amino acid is electrostatically interacting with the polymer; wherein the metal oxide comprises metal ions in a 3+ and 4+ valence state; and wherein the metal oxide is selected from lanthanide oxide and / or zirconium oxide.

[0011] In some embodiments, the lanthanide oxide comprises a lanthanide, the lanthanide selected from La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, or a combination thereof.

[0012] In some embodiments, the metal oxide is characterised by a 4+ to 3+ valence ratio of about 0.2 to about 0.7.

[0013] In some embodiments, the metal ion with a 4+ valence is characterised by a mass percentage about 15% to about 30% relative to the metal oxide nanoparticle.

[0014] In some embodiments, the amino acid is a L amino acid, D amino acid or a combination thereof.

[0015] In some embodiments, the amino acid is selected from D-arginine (D-Arg), L- arginine (L-Arg), D-methionine (D-Met), L-methionine (L-Met), D-tyrosine (D- Tyr), L-tyrosine (L-Tyr), or a combination thereof.

[0016] In some embodiments, the polymer is selected from polyacrylic acid (PAA), Dextra, Guar gum, Gelatin, Xanthan gum, Carboxymethyl cellulose sodium,

[0017] Carboxymethyl starch (sodium), Crosslinked polyacrylate / C10-30 alkyl acrylate copolymer, Acrylate / C16-20 alkyl ethoxylate (20) cocopolymer, Acrylate / C16- 20 alkyl ethoxylate (20) methacrylate copolymer, Acrylate / C18 alkyl ethoxylate (20) methacrylate copolymer, Acrylate / VA copolymer, Sodium acrylate / ethylene / acrylic acid copolymer, Carbomer, and their derivatives thereof.

[0018] In some embodiments, the polymer is a polyelectrolyte.

[0019] In some embodiments, the polymer is characterised by a molecular weight of about 500 to about 5000.

[0020] In some embodiments, the amino acid is D-arginine and the polymer is polyacrylic acid.

[0021] In some embodiments, the amino acid is configured to interact with metal oxide through coordination to the metal ion via its amino moiety.

[0022] In some embodiments, the polymer is configured to interact with metal oxide through coordination to the metal ion via its ionisable moiety.

[0023] In some embodiments, the weight ratio of amino acid to polymer is about 75:25.

[0024] In some embodiments, a mole ratio of metal oxide to (amino acid and polymer) is about 1 : 1 to about 1 : 10.

[0025] In some embodiments, the metal oxide nanoparticle comprises a corona, the corona comprising a protein, a surfactant, a ligand, or a combination thereof.

[0026] In some embodiments, the metal oxide nanoparticle is characterised by an aqueous dispersibility for at least 30 days.

[0027] In some embodiments, the metal oxide nanoparticle is attached to a probiotic.

[0028] The present disclosure provides an enzyme-probiotic conjugate, comprising the metal oxide nanoparticle as disclosed herein conjugated to a probiotic.

[0029] In some embodiments, the probiotic is selected from Clostridium butyricum, Bifidobacterium, Lactobacillus, Komagataeibacter, Leuconostoc, or a combination thereof.

[0030] In some embodiments, the number ratio of enzyme to probiotic is about 1 to about 5000.

[0031] The present disclosure provides a use of the metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein as an apyrase mimic and / or a superoxide dismutase mimic.

[0032] The present disclosure provides a method of fabricating a metal oxide nanoparticle, comprising mixing a metal salt, polyelectrolyte and amino acid with a base at ambient temperature.

[0033] The present disclosure also provides a method of treating an inflammatory disease and / or a wound in a subject in need thereof, comprising administering a therapeutic amount of the metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein to the subject.

[0034] The present disclosure provides a metal oxide nanoparticle or the enzymeprobiotic conjugate as disclosed herein for use in treating an inflammatory disease and / or a wound.

[0035] The present disclosure provides use of a metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein in the manufacture of a medicament for the treatment of an inflammatory disease and / or a wound.

[0036] In some embodiments, the inflammatory disease is characterised by an elevated extracellular ATP release relative to a control.

[0037] In some embodiments, the inflammatory disease is selected from inflammatory bowel disease (IBD), osteoarthritis, stroke, acute kidney injury, acute liver injury, myocardial infarction, colitis, ulcerative colitis, and Crohn's disease.

[0038] Brief description of the drawings

[0039] Embodiments of the present invention will now be described, by way of nonlimiting example, with reference to the drawings in which :

[0040] Figure 1. Schematic preparation of the amino acid modulated CeO2apyrasemimic and their application in inflammatory treatment.

[0041] Figure 2. (a) Transmission electron microscopy (TEM) of water soluble PAA- CeO2, 25%LA-CeO2, 50%LA-CeO2, 75%LA-CeO2, 25%DA-CeO2, 50%DA-CeO2, and 75%DA-CeO2.

[0042] Figure 3. (a) X-ray Diffraction (XRD) pattern of PAA-CeO2, 25%LA-CeO2, 50° / ol_A-CeO2, 75%LA-CeO2, 25%DA-CeO2, 50%DA-CeO2, and 75%DA-CeO2and (b) Fourier Transform Infrared (FTIR.) spectrum of PAA, L-Arg, D-Arg, PAA- CeO2, 25%LA-CeO2, 50%LA-CeO2, 75%LA-CeO2, 25%DA-CeO2, 50%DA-CeO2, and 75%DA-CeO2. scavenging ability of different concentration of 75%LA-CeO2 and 75%DA-CeO2. Data are presented as mean ± s.d. from n = 3 biological replicates.

[0043] Figure 6. HPLC results of catalytic hydrolysis of ATP molecules by PAA-CeO2, 25° / oLA-CeO2, 50%LA-CeO2, 75%l_A-CeO2, 25%DA-CeO2, 50° / oDA-CeO2, and 75%DA-CeO2, as well as their apyrase-like catalytic scheme.

[0044] Figure 7. ATP-scavenging ability of other traditional apyase-like nanozymes. Data are presented as mean ± s.d. from n = 3 biological replicates.

[0045] Figure 8. ATP-scavenging ability of natural apyase after undergoing SCF, SIF and SCF tested in the (a) first time and (b) second time. Data are presented as mean ± s.d. from n = 3 biological replicates.

[0046] Figure 9. (a) SOD-like, (b) CAT-like, (c) OXD-like and (d) POD-like ability of PAA-CeO2, 25%LA-CeO2, 50%U\-CeO2, 75%LA-CeO2, 25%DA-CeO2, 50%DA- CeO2, and 75%DA-CeO2. Data are presented as mean ± s.d. from n = 3 biological replicates.

[0047] Figure 10. The large-scale production of 75%DA-CeO2.

[0048] Figure 11. Pictures of 75%DA-CeO2dispersed in H2O over consecutive days.

[0049] Figure 12. ATP-scavenging ability of 75%DA-CeO2dispersed in H2O over consecutive days. Data are presented as mean ± s.d. from n = 3 biological replicates.

[0050] Figure 13. (a) high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and (b) TEM images and (c) mapping of CB@DA.

[0051] Figure 14. (a) ATP-consuming ability of 75%DA-CeO2, CB, CB+75%DA-CeO2and CB@75%DA-CeO2with equal concentration in HT29 cell. 12P, 13P, 14P and 15P indicate 75%DA-CeO2, CB, CB+75%DA-CeO2and CB@75%DA-CeO2versus Control group, respectively, (b) ATP-consuming ability of CB@75%DA-CeO2with different concentration in buffer, 12P, 13P, 14P and 15P indicate 16.5 ug / mL, 33 ug / mL, 66 ug / mL, 132 ug / mL versus the 0 ug / mL, respectively. Data are presented as mean ± s.d. from n = 3 biological replicates.

[0052] Figure 15. (a) ATP-consuming ability of 75%DA-CeO2, CB, CB+75%DA-CeO2and CB@75%DA-CeO2with equal concentration in HT29 cell. 12P, 13P, 14P and 15P indicate 75%DA-CeO2, CB, CB+75%DA-CeO2and CB@75%DA-CeO2versus Control group, respectively, (b) ATP-consuming ability of CB@75%DA-CeO2with different concentration in HT29 cell, 12P, 13P, 14P and 15P indicate 16.5 ug / mL, 33 ug / mL, 66 ug / mL, 132 ug / mL versus the 0 ug / mL, respectively. Data are presented as mean ± s.d. from n = 3 biological replicates.

[0053] Figure 16. ATP-scavenging abilities of CB@75%DA-CeO2and CB in simulated GI environments of SGF, SIF and SCF. Data are presented as mean ± s.d. from n = 3 biological replicates.

[0054] Figure 17. Corresponding viability of BL in SGF, SIF and SCF. Data are presented as mean ± s.d. from n = 3 biological replicates.

[0055] Figure 18. Antioxidant artificial enzymes-armed CB probiotics ameliorate TNBS-induced CD. (a) Daily body weight changes in each group for 12 days. Data were normalized as a percentage of the body weight at day 0. (b) Colon lengths of mice with indicated treatments on day 8. (c) Representative photographs of colons of mice on day 8 after indicated treatments, respectively. Data are presented as mean ± s.d. from n = 5 biological replicates. 21P, 23P, 24P, 25P and 26P indicate that Health, 75%DA-CeC>2, CB, CB+75%DA-CeC>2 and CB@75%DA-CeO2 versus the control group.

[0056] Figure 19. (a) Colon ATP, (b) fecal ATP, (c) colon IL-18 and (d) colon IL-6 of DC mice after different treatment. Data are presented as mean ± s.d. from n = 6 biological replicates. 21P, 23P, 24P, 25P and 26P indicate that Health, 75%DA- CeO2, CB, CB+75%DA-CeO2 and CB@75%DA-CeO2 versus the control group.

[0057] Figure 20. Comparison of CD therapy between CB@75%DA-CeO2 and clinical therapy. Data are presented as mean ± s.d. from n = 5 biological replicates. 12P, 13P and 14P indicate that anti-TNF o, anti-a4£7 and CB@75%DA-CeO2 versus the Control group.

[0058] Figure 21. Antioxidant artificial enzymes-armed CB probiotics ameliorate DSS- induced UC. (a) Daily body weight changes in each group for 9 days. Data were normalized as a percentage of the body weight at day 0. (b) Colon lengths of mice with indicated treatments on day 8. (c) Representative photographs of colons of mice on day 8 after indicated treatments, respectively. 21 P, 23P, 24P, 25P and 26P indicate that Health, 75%DA-CeO2, CB, CB+75%DA-CeO2and CB@75%DA-CeO2 versus the control group.

[0059] Figure 22. Hematological analysis of the healthy mice or CD mice after different therapy. Data are presented as mean ± s. d. from n = 3 biological replicates.

[0060] Figure 23. Blood biochemical analysis of the healthy mice or CD mice after different therapy. Data are presented as mean ± s. d. from n = 3 biological replicates.

[0061] Figure 24. Hematological analysis of the healthy mice or UC mice after different therapy. Data are presented as mean ± s. d. from n = 3 biological replicates. Figure 25. Blood biochemical analysis of the healthy mice or UC mice after different therapy. Data are presented as mean ± s. d. from n = 3 biological replicates.

[0062] Figure 26. Body weights of the healthy mice orally administered with or without CB@75%DA-CeO2 during 28 days. Data are presented as mean ± s. d. from n = 3 biological replicates.

[0063] Figure 27. Biosafety evaluation of CB@75%DA-CeO2 through hematological analysis. Healthy mice were orally administered with or without CB@75%DA- CeOz for 28 days, and their blood samples were collected for serum biochemistry assay. Data are presented as mean ± s. d. from n =3 biological replicates.

[0064] Figure 28. Biosafety evaluation of CB@75%DA-CeO2 through biochemical analysis. Healthy mice were orally administered with or without CB@75%DA- CeC>2 for 28 days, and their blood samples were collected for serum biochemistry assay. Data are presented as mean ± s. d. from n =3 biological replicates.

[0065] Figure 29. X-ray Diffraction (XR.D) pattern of PAA-CeC , 25%l_A-CeO2, 50%LA- CeO2, 75%LA-CeO2, 25%DA-CeO2, 50%DA-CeO2, and 75%DA-CeO2.

[0066] Figure 30. Treatment of monosodium iodoacetate (MIA)-induced osteoarthristis (OA) rats with DA-CeC>2 nanoparticle relative to control.

[0067] Figure 31. The o-diversity of faecal microbiome illustrated by Shannon's index (a), p-diversity of faecal microbiome demonstrated by non-metric multidimensional scaling (NMDS) plot (b) and relative abundance of gut microbiome illustrated at the family-level taxonomy after different treatments (c). Family-level taxonomy is presented as a percentage of the total sequences (d-f).

[0068] Detailed description

[0069] The present disclosure provides a metal oxide nanoparticle comprising: a) metal oxide, the metal capable of being a Lewis acid; b) a polymer comprising an ionisable moiety; and c) an amino acid; wherein a weight ratio of amino acid to polymer is about 0.1 :99.9 to about 99.9:0.1; and wherein the amino acid is electrostatically interacting with the polymer.

[0070] The metal oxide may be a lanthanide oxide, or may be zirconium oxide. These metals exhibit strong Lewis acid characteristics and has a high affinity for ATP.

[0071] Accordingly, in some embodiments, the metal oxide nanoparticle comprises: a) a metal oxide, the metal capable of being a Lewis acid; b) a polymer comprising an ionisable moiety; and c) an amino acid; wherein a weight ratio of amino acid to polymer is about 0.1 :99.9 to about 99.9:0.1; wherein the amino acid is electrostatically interacting with the polymer; and wherein the metal oxide is selected from lanthanide oxide and / or zirconium oxide.

[0072] The present disclosure provides a lanthanide oxide nanoparticle or zirconium oxide nanoparticle, comprising: a) lanthanide oxide or zirconium oxide; b) a polymer comprising an ionisable moiety; and c) an amino acid; wherein a weight ratio of amino acid to polymer is about 0.1 :99.9 to about 99.9:0.1; and wherein the amino acid is electrostatically interacting with the polymer.

[0073] The metal oxide nanoparticle is a cluster nanoparticle. IUPAC defines "cluster" as "a number of metal centers grouped close together which can have direct metal bonding interactions or interactions through a bridging ligand, but are not necessarily held together by these interactions". Nanoclusters (NCs) are thus clusters in the nanometre size that may be stabilized with peripheral protective groups. The cluster core of the nanocluster is usually less than 2 nm. However, with the protective groups, the overall size of the nanocluster may be larger than 2 nm. In the present context, the polymer and amino acid may act as a bridging ligand, and may also act as a stabilising peripheral ligand.

[0074] The metal oxide nanoparticle is water soluble, and may act as nanozymes. In particular, the metal oxide acts as a potent Lewis acid with a strong affinity for substrates, thus serving as the catalytic center by binding with phosphate ions to degrade ATP. The polymer enhances the stability and aqueous dispersibility of the ultra-small nanoparticle. When the amino acid is not covalently bonded to polyelectrolyte, the amino acid exhibits a robust electron-donating capability. This modulates the metal oxide's valence state, promoting a higher prevalence of tetravalent metal.

[0075] While lanthanides may exist in valence states of 2, 3 or 4, the 3+ valence state is the most stable. Without wanting to be bound by theory, the inventors have found that the interaction of amino acid with PAA may stabilise the 4+ valence state of lanthanide, thus preferentially forming LnOz instead of LnzOs. This is confirmed with XRD characterization, that the Ce-based lanthanide oxide preferentially forms CeOz rather than CezOs (Figure 29).

[0076] In some embodiments, the lanthanide oxide comprises a lanthanide, the lanthanide selected from La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, or a combination thereof. In some embodiments, the lanthanide oxide comprises a lanthanide, the lanthanide selected from La, Ce, or a combination thereof. In some embodiments, the lanthanide is Ce.

[0077] In some embodiments, the metal oxide comprises a metal ion, wherein the metal ion is in a 4+ valence state. The lanthanide oxide may be LnOz. In some embodiments, the metal oxide comprises a metal ion, wherein the metal ion is in a 3+ valence state. The lanthanide oxide may be LnzCh. In some embodiments, the metal oxide comprises a metal ion, wherein the metal ion is in a 3+ and 4+ valence state.

[0078] In some embodiments, the metal oxide is characterised by a 4+ to 3+ valence ratio of about 0.2 to about 0.7. In some embodiments, the valence ratio is about 0.3 to about 0.7, about 0.4 to about 0.7, or about 0.4 to about 0.6. In some embodiments, the valence ratio is about 0.6.

[0079] In some embodiments, the metal ion with a 4+ valence is characterised by a mass percentage about 15% to about 30% relative to the metal oxide nanoparticle. The mass percentage may be characterized by XPS. In some embodiments, the metal ion with a 4+ valence is characterised by a mass percentage about 18% to about 30% relative to the metal oxide nanoparticle, about 18% to about 25%, about 20% to about 25%, or about 20% to about 21%.

[0080] In some embodiments, the metal oxide nanoparticle comprises a core, wherein the core comprises the metal oxide. The metal oxide may form clusters within the core.

[0081] Amino acids are rich in nitrogen, which endows them with a strong electrondonating capability. This characteristic significantly influences the valence state of cerium (Ce), leading to a greater prevalence of tetravalent cerium.

[0082] In some embodiments, the amino acid is a L amino acid, D amino acid or a combination thereof. L amino acids are amino acids where the stereogenic carbon alpha to the amino group has the L-configuration. L amino acids are mostly naturally occurring amino acids. D amino acids are amino acids where the stereogenic carbon alpha to the amino group has the D-configuration. L- and D-amino acids are usually enantiomers. In some embodiments, the amino acid is a D amino acid.

[0083] In some embodiments, the amino acid is selected from arginine, methionine, tyrosine, or a combination thereof. In some embodiments, the amino acid is selected from D-arginine (D-Arg), L-arginine (L-Arg), D-methionine (D-Met), L- methionine (L-Met), D-tyrosine (D-Tyr), L-tyrosine (L-Tyr), or a combination thereof. In some embodiments, the amino acid is selected from D-arginine (D- Arg), L-arginine (L-Arg), or a combination thereof.

[0084] In some embodiments, the metal oxide nanoparticle is 75% DA-CeCh. This nanoparticle comprises CeC>2, PAA and D-arginine. The mass ratio of D-arginine to PAA is 75:25. In this regard, the mass of D-arginine is about 3 times relative to PAA. Based on this ratio, the mass percentage of Ce4+is about 20% to about 21%, as characterized by XPS.

[0085] In particular, it was found that arginine improves the ATP regulation and thus enhance apyrase-like activity. Further, it was found that D-Arg has superior ATP-scavenging activity relative to other amino acids. It is believed that the interaction between D-arginine and CeO2 facilitates the formation or stabilization of Ce4+active sites, which may enhance the material's ATP- scavenging capability. However, further studies are needed to confirm the underlying mechanism.

[0086] In some embodiments, the metal oxide nanoparticle comprises a core, wherein the core comprises the amino acid. In some embodiments, the core comprises metal oxide and amino acid.

[0087] The amino acid may modulate the structure of the metal oxide cluster within the core. For example, the metal oxide cluster may have crystallographic planes corresponding to (111), (220), and (222).

[0088] In some embodiments, the polymer is selected from polyacrylic acid (PAA), Dextra, Guar gum, Gelatin, Xanthan gum, Carboxymethyl cellulose sodium, Carboxymethyl starch (sodium), Crosslinked polyacrylate / C10-30 alkyl acrylate copolymer, Acrylate / C16-20 alkyl ethoxylate (20) cocopolymer, Acrylate / C16- 20 alkyl ethoxylate (20) methacrylate copolymer, Acrylate / C18 alkyl ethoxylate

[0089] (20) methacrylate copolymer, Acrylate / VA copolymer, Sodium acrylate / ethylene / acrylic acid copolymer, Carbomer, and their derivatives thereof.

[0090] The polymer may comprise an ionisable moiety. For example, the ionisable moiety may be on a pendant chain or forms a pendant group on the polymer. Thus, when solvated in an aqueous solution with a high or low pH, the ionisable moieties on the polymer may be ionised to give negative or positive charges respectively. Counterions may further associate with the ionised polymer in order to balance the charges. Examples of ionisable moieties include, but is not limited to, carboxylate, amine, hydroxy, sulfonate, or a combination thereof.

[0091] In some embodiments, the polymer is a polyelectrolyte. Examples of polyelectrolytes include, but are not limited to, polyacrylic acid (PAA), poly(styrene sulfonate) (PSS), polyethyleneimine, polyallylamine, polyacrylamide, poly(vinyl alcohol), poly(dimethylaminopropylacrylmide, or a combination thereof.

[0092] In some embodiments, the polymer is characterised by a molecular weight of about 500 to about 5000. In other embodiments, the molecular weight is about 800 to about 5000, about 1000 to about 5000, about 1500 to about 5000, about 2000 to about 5000, about 2500 to about 5000, about 3000 to about 5000, about 3500 to about 5000, or about 4000 to about 5000.

[0093] The amino acid is electrostatically interacting with the polymer or polyelectrolyte. In this regard, the amino acid is not covalently bonded to the polymer or polyelectrolyte. Amino acid and polymer or polyelectrolyte are linked to the lanthanide oxide through the coordination modes of Lanthanide-N and Lanthanide-O, respectively. The metal oxide thus forms clusters within the core, which may be separately and away from each other by the polymer.

[0094] In some embodiments, the metal oxide nanoparticle comprises a core, wherein the core comprises the polymer. In some embodiments, the core comprises metal oxide and polymer. In some embodiments, the core comprises polymer and amino acid. In some embodiments, the core comprises metal oxide, amino acid, and polymer.

[0095] Without wanting to be bound by theory, it is believed that lanthanides are strong Lewis acids that coordinate with hard bases (carboxylates) and highly electronegative donors such as N or 0. For example, the lanthanide may coordinate with the guanidinyl moiety of arginine, or the oxyacyl moiety of PAA. From XPS analysis of various CeOz samples, it is evident that the presence of more arginine (Arg) correlates with a higher abundance of Ce(IV). This suggests that Arg influences the coordination of Cerium ions. It is thus hypothesized that Ce(IV) is coordinated with both Ce-N and Ce-0 bonds.

[0096] In some embodiments, the amino acid is configured to interact with metal oxide through coordination to the metal ion via its amino moiety. In some embodiments, the polymer is configured to interact with metal oxide through coordination to the metal ion via its ionisable moiety (such as acyl and / or oxy moiety). The ionisable moiety may be ionised to further improve its interaction.

[0097] "Amino" refers to the group -NR"R" where each R" is independently hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl and where each of alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl is as described herein.

[0098] "Oxo / hydroxy" refers to groups =0, H0-.

[0099] "Acyl" refers to groups H-C(O)-, alkyl-C(O)-, cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclyl-C(O)-, where alkyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are as described herein.

[0100] In some embodiments, the metal oxide nanoparticle is at least partially surface passivated by the polymer. In this regard, the polymer forms a corona on the metal oxide nanoparticle. In some embodiments, the metal oxide nanoparticle is at least partially surface passivated by the amino acid. In some embodiments, the metal oxide nanoparticle is at least partially surface passivated by the polymer and the amino acid. In some embodiments, the metal oxide nanoparticle is at least partially surface passivated by the metal oxide. In some embodiments, the metal oxide nanoparticle is at least partially surface passivated by the metal oxide and the polymer. In some embodiments, the metal oxide nanoparticle is at least partially surface passivated by the metal oxide and the amino acid. In some embodiments, the metal oxide nanoparticle is at least partially surface passivated by the metal oxide, the polymer and the amino acid.

[0101] In some embodiments, the weight ratio of amino acid to polymer is about 0.1:99.9 to about 99.9:0.1, about 1:99 to about 99:1, about 10:90 to about 99:1, about 15:85 to about 99:1, about 20:80 to about 99:1, about 25:75 to about 99:1, about 30:70 to about 99:1, about 35:65 to about 99:1, about 40:60 to about 99:1, about 45:55 to about 99:1, about 50:50 to about 99:1, about 55:45 to about 99:1, about 60:40 to about 99:1, about 65:35 to about 99:1, about 70:30 to about 99:1, about 75:25 to about 99:1, about 80:20 to about 99:1, about 85:15 to about 99:1, or about 90:10 to about 99:1. In some embodiments, the weight ratio of amino acid to polymer is about 75:25. It should be noted that the weight ratio of amino acid to polymer refers to their content in the metal oxide nanoparticle, which includes the corona of the metal oxide nanoparticle (if present).

[0102] In some embodiments, a mole ratio of metal oxide to (amino acid and polymer) is about 1:1 to about 1:10. In other embodiments, the mole ratio is about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, or about 1:2 to about 1:7.

[0103] In some embodiments, the metal oxide nanoparticles comprises a corona. The corona is formed by surface functionalising the metal oxide nanoparticles. The corona may further comprise a protein, a surfactant, a ligand, or a combination thereof. For example, bovine serum albumin may be used to further improve the aqueous dispersibility of the metal oxide nanoparticles. Charged surfactants and / or ligands may be used to further improve the aqueous dispersibility of the metal oxide nanoparticles. These entities may either be covalently bonded to the metal oxide nanoparticles, or electrostatically interacting with the metal oxide nanoparticles.

[0104] In some embodiments, the metal oxide nanoparticle comprises a core and a corona. In some embodiments, the core comprises metal oxide and the corona comprises amino acid and polymer. In some embodiments, the core comprises metal oxide, and the corona comprises metal oxide, amino acid and polymer.

[0105] In some embodiments, the core comprises metal oxide and amino acid, and the corona comprises polymer. In some embodiments, the core comprises metal oxide and amino acid, and the corona comprises amino acid and polymer. In some embodiments, the core comprises metal oxide and amino acid, and the corona comprises metal oxide and polymer. In some embodiments, the core comprises metal oxide and amino acid, and the corona comprises metal oxide, amino acid and polymer.

[0106] In some embodiments, the core comprises metal oxide, amino acid and polymer, and the corona comprises amino acid. In some embodiments, the core comprises metal oxide, amino acid and polymer, and the corona comprises polymer. In some embodiments, the core comprises metal oxide, amino acid and polymer, and the corona comprises metal oxide. In some embodiments, the core comprises metal oxide, amino acid and polymer, and the corona comprises metal oxide and amino acid. In some embodiments, the core comprises metal oxide, amino acid, and polymer, and the corona comprises metal oxide and polymer. In some embodiments, the core comprises metal oxide, amino acid and polymer, and the corona comprises amino acid and polymer. In some embodiments, the core comprises metal oxide, amino acid and polymer, and the corona comprises metal oxide, amino acid and polymer.

[0107] In some embodiments, the core comprises amino acid, and the corona comprises metal oxide and polymer. In some embodiments, the core comprises amino acid, and the corona comprises metal oxide, amino acid and polymer. In some embodiments, the core comprises polymer, and the corona comprises metal oxide and amino acid. In some embodiments, the core comprises polymer, and the corona comprises metal oxide, amino acid and polymer.

[0108] In some embodiments, the metal oxide nanoparticle is characterised by an aqueous dispersibility. The metal oxide nanoparticles may exhibit stability in water owing to their encapsulation and stabilization by the polymer such as PAA.

[0109] In some embodiments, the metal oxide nanoparticle is characterised by a stability in an aqueous medium of at least 30 days.

[0110] The term 'aqueous medium' used herein refers to a water based solvent or solvent system, and which comprises of mainly water. Such solvents can be either polar or non-polar, and / or either protic or aprotic. Solvent systems refer to combinations of solvents which resulting in a final single phase. Both 'solvents' and 'solvent systems' can include, and is not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethylether, dichloromethane, tetra hydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol or water. Water based solvent or solvent systems can also include dissolved ions, salts and molecules such as amino acids, proteins, sugars and phospholipids. Such salts may be, but not limited to, sodium chloride, potassium chloride, ammonium acetate, magnesium acetate, magnesium chloride, magnesium sulfate, potassium acetate, potassium chloride, sodium acetate, sodium citrate, zinc chloride, HEPES sodium, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate and sodium phosphate. As such, biological fluids, physiological solutions and culture medium also falls within this definition.

[0111] In some embodiments, the metal oxide nanoparticle comprises: a) at least one CeO? cluster; b) polyacrylic acid (PAA); and c) arginine; wherein a weight ratio of arginine to PAA is about 75:25; wherein the amino acid is electrostatically interacting with the polymer.

[0112] In some embodiments, the metal oxide nanoparticle comprises: a) at least one CeCh cluster; b) polyacrylic acid (PAA); and c) D-arginine; wherein a weight ratio of D-arginine to PAA is about 75:25; wherein the amino acid is electrostatically interacting with the polymer.

[0113] In some embodiments, the metal oxide nanoparticle is attached to a probiotic.

[0114] A probiotic refers to live microorganisms, such as bacteria and / or yeast, that provide health benefits when consumed, particularly by improving gut health. Probiotics are often used to maintain and / or restore gut health.

[0115] The metal oxide nanoparticle may be attached to a surface of the probiotic. For example, the metal may be coordinated to polysaccharides and / or phosphate groups present on the bacterial cell wall. This coordination may be non-covalent interaction. This increases the retention time of lanthanide oxide nanoparticle in a body of a subject.

[0116] In some embodiments, the interaction between metal oxide nanoparticles and polysaccharides and / or phosphate groups is coordinate-covalent bonding.

[0117] Accordingly, the present disclosure provides an enzyme-probiotic conjugate, comprising the metal oxide nanoparticle as disclosed herein conjugated to a probiotic. The present disclosure provides a composition comprising the metal oxide nanoparticle as disclosed herein conjugated to a probiotic. The composition may be a probiotic composition. The metal oxide nanoparticle acts as an artificial enzyme (or apyrase mimic) in the sense that it acts like apyrase in its scavenging ability.

[0118] It was found that the enzyme-probiotic conjugate is more stable over various environmental conditions compared to a use of a protein enzyme. This allows the enzyme-probiotic conjugate to demonstrate good efficacy, and also shelf life.

[0119] In some embodiments, the probiotic is selected from Clostridium butyricum, Bifidobacterium, Lactobacillus, Komagataeibacter, Leuconostoc, or a combination thereof. In some embodiments, the probiotic is selected from Clostridium butyricum, Lactobacillus, or a combination thereof. Clostridium butyricum and Lactobacillus may tolerate acidic stomach to some extent and hence enabling greater colonization in the intestine and enhancing its therapeutic effects on IBD.

[0120] In some embodiments, the number ratio of enzyme to probiotic is about 1 to about 5000. The number ratio refers to the number of metal oxide nanparticles attached to a single probiotic. In other embodiments, the number ratio is about 1 to about 4500, about 1 to about 4000, about 1 to about 3500, about 1 to about 3000, about 1 to about 2500, about 1 to about 2000, about 1 to about 1500, about 1 to about 1000, about 1 to about 500, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 1 to about 80, or about 1 to about 50.

[0121] The present disclosure provides a use of the metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein as an apyrase mimic. In this regard, similar to apyrase, the metal oxide nanoparticle hydrolyses ATP to yield

[0122] AMP and inorganic phosphate. It is believed that the metal oxide nanoparticle scavenges ATP owing to the robust binding affinity between metal ions and the phosphate group of ATP.

[0123] In some embodiments, the metal oxide nanoparticle is characterised by a ATP scavenging ability. The ATP scavenging ability may be determined using ATP kits, with the HEPES buffer serving as the control.

[0124] The present disclosure also provides a use of the metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein as superoxide dismutase mimic. In this regard, the metal oxide nanoparticle causes the dismutation (or partitioning) of the superoxide (O2 ) radical into ordinary molecular oxygen (O2) and hydrogen peroxide (H2O2).

[0125] In some embodiments, the metal oxide nanoparticle is characterised by a superoxide dismutation (SOD) ability. The SOD scavenging ability may be determined using SOD kits, with the HEPES buffer serving as the control.

[0126] In some embodiments, the present disclosure provides water-soluble cerium oxide nanozymes with attributes conducive to scalable production, showcasing pronounced ATP-clearance capabilities. The introduction of arginine has been demonstrated to augment the concentration of tetravalent cerium, consequently elevating its catalytic activity. These nanozymes manifest the ability to sequester extracellular ATP (eATP) abundantly released during inflammatory processes by damaged or deceased cells. By intercepting the ATP-P2X7 purinergic signaling, these nanozymes exhibit robust and broad-spectrum antiinflammatory properties, substantiating their efficacy across diverse pathological conditions. Finally, in order to achieve sustained treatment for inflammatory bowel disease (IBD), CeCh is tethered onto the surface of probiotics, enhancing the retention of CeC in the intestines and facilitating continuous antioxidant therapy.

[0127] The present disclosure provides a method of fabricating a metal oxide nanoparticle as disclosed herein, comprising mixing a metal salt, polyelectrolyte and amino acid with a base. The mixing may occur for at least 5 h, or at least 10 h. The method may be conducted at ambient temperature.

[0128] The present disclosure provides a pharmaceutical composition comprising the metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein.

[0129] The present disclosure also provides a method of treating an inflammatory disease in a subject in need thereof, comprising administering a therapeutic amount of the metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein to the subject.

[0130] The present disclosure provides a metal oxide nanoparticle or the enzymeprobiotic conjugate as disclosed herein for use in treating an inflammatory disease.

[0131] The present disclosure provides use of a metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein in the manufacture of a medicament for the treatment of an inflammatory disease.

[0132] In some embodiments, the inflammatory disease is characterised by an elevated extracellular ATP release relative to a control. The control may be a nondiseased cell, or a cell derived from a comparable organ of a healthy subject.

[0133] In some embodiments, the inflammatory disease is selected from inflammatory bowel disease (IBD), osteoarthritis, stroke, acute kidney injury, acute liver injury, myocardial infarction, colitis, ulcerative colitis, and Crohn's disease.

[0134] The present disclosure also provides a method of treating a wound in a subject in need thereof, comprising administering a therapeutic amount of the metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein to the subject.

[0135] The present disclosure provides a metal oxide nanoparticle or the enzymeprobiotic conjugate as disclosed herein for use in treating a wound.

[0136] The present disclosure provides use of a metal oxide nanoparticle or the enzyme-probiotic conjugate as disclosed herein in the manufacture of a medicament for the treatment of a wound.

[0137] The nanoparticle of the invention can be administered to a subject as a pharmaceutically acceptable salt thereof. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

[0138] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. In particular, the present invention includes within its scope cationic salts eg sodium or potassium salts, or alkyl esters (eg methyl, ethyl) of the phosphate group.

[0139] Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.

[0140] The nanoparticle of the invention may be in crystalline form or as a solvate (e.g. hydrate) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.

[0141] The nanoparticle of the invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof is administered to the patient in a therapeutically effective amount. As used herein, a therapeutically effective amount is intended to include at least partially attaining the desired effect, or delaying the onset of, or inhibiting the progression of, or halting or reversing altogether the onset or progression of inflammation.

[0142] The term "therapeutic effect" refers to some extent of relief of one or more of the symptoms of a disorder (e.g., a neoplasia or tumor) or its associated pathology. "Therapeutically effective amount" as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. "Therapeutically effective amount" is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the "therapeutically effective amount" (e.g., ED50) of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0143] As used herein, the term "effective amount" relates to an amount of compound which, when administered according to a desired dosing regimen, provides the desired therapeutic activity. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods.

[0144] Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage.

[0145] Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.

[0146] The compound of the invention may be administered in a single dose or a series of doses. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

[0147] The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0148] The nanoparticles may be administered via topical or intravenous administration. For example, topical application typically involves administering the compound of the invention in an amount between 0.1 ng and 10 mg.

[0149] The compound, composition or combinations of the invention may also be suitable for intravenous administration. For example, the nanoparticle or a pharmaceutically acceptable salt, solvate or prodrug thereof may be administered intravenously at a dose of up to 16 mg / m2.

[0150] The nanoparticle or composition of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or nonaqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. In another embodiment, the nanoparticle or a pharmaceutically acceptable salt, solvate or prodrug is orally administerable.

[0151] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

[0152] The nanoparticle or composition of the invention may be suitable for topical administration in the mouth including lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0153] The nanoparticle or composition of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the compounds of the invention.

[0154] The nanoparticle or composition of the invention may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the compound, composition or combination isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compound, composition or combination may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze- dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0155] Preferred unit dosage composition are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the active ingredient.

[0156] It should be understood that in addition to the active ingredients particularly mentioned above, the composition or combination of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and / or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

[0157] The compositions (e.g., pre-formulations, formulations) may be prepared using conventional methods that are well known in the field of pharmacy. For example, methods that use standard laboratory or pharmaceutical processing equipment are well known to those in the field of pharmacy.

[0158] For example, the composition may be a dry powder composition, and may be reconstituted, diluted, hydrated, re-hydrated to a form which is suitable for use. For example, a final composition may be prepared from a dry powder composition, for example, extemporaneously, by dilution, reconstitution, hydration, re-hydration, etc., using appropriate liquids, e.g., water (e.g., water- for-injection), aqueous saline (e.g., 0.9% w / v saline solution), aqueous glucose (e.g., 5% w / v glucose solution), etc. The dry powder composition may thus comprise lipholised, dehydrated, freeze-dried, and / or spray dried nanoparticles (and / or probiotics).

[0159] Examples

[0160] The lanthanide oxide nanoparticles comprises three components: a) Lanthanides such as Ce4+, Zr4+, La3+, which acts as a potent Lewis acid with a strong affinity for substrates, thus serving as the catalytic center by binding with phosphate ions to degrade ATP; b) polyacrylic acid (PAA), which enhances the stability and aqueous dispersibility of the ultra-small CeO2structure; and c) amino acids, such as D-arginine (D-Arg), L-arginine (L-Arg), D- methionine (D-Met), L-methionine (L-Met), D-tyrosine (D-Tyr), L- tyrosine (L-Tyr), and others containing nitrogen, exhibiting a robust electron-donating capability. This contribution significantly modulates the lanthanide's (such as cerium) valence state, promoting a higher prevalence of tetravalent cerium.

[0161] A general protocol is as follows: Dropwise add 5 mL of a mixed solution containing different ratios of Ce(NO3)3'6H2O, polyacrylic acid (PAA), and D- arginine / L-arginine into 15 mL of ammonia solution. After reacting at room temperature (such as from 12 °C to 40 °C) for 24 hours, collect the solution, dialyze, and then freeze-dry.

[0162] In some embodiments, Ce4+was utilised along with D-Arg and L-Arg as the respective catalytic center and regulator. Ce4+exhibits strong Lewis acidity, while Arginine (Arg) is rich in electron-donating nitrogen, enhancing ATP- hydrolysis ability. The outcome of the approach is the successful production of a well-dispersed ultra-small CeO2enriched in tetravalent cerium. Notably, D- Arg regulation enhances ATP clearance capability. Additionally, the CeO2demonstrates the ability to scavenge superoxide radicals (O2. ), with this capability unaffected by D-Arg. The D-Arg modified CeO2exhibits robust ATP- scavenging activity compared to traditional nanozymes, coupled with ultrastable performance relative to natural apyrase.

[0163] The lanthanide nanoparticles is suitable for treating inflammatory diseases, particularly those involving elevated extracellular ATP (eATP) release from damaged cells. Excess eATP activates eATP-P2X7 receptor signaling, triggering inflammation in conditions such as inflammatory bowel disease (IBD), osteoarthritis, stroke, acute kidney injury, acute liver injury, myocardial infarction, wound healing, etc.

[0164] Arginine regulates the structure of CeO?

[0165] Leveraging the robust binding affinity between lanthanide ions and the phosphate group of ATP, lanthanide nanoparticles could effectively emulate apyrase function to sequester ATP. In our initial investigation, we successfully synthesized varying ratios of arginine-regulated cerium oxide denoted as PAA- CeO2, 25%LA-CeO2, 50%LA-CeO2, 75%LA-CeO2, 100%LA-CeO2, 25%DA-CeO2, 50%DA-CeO2, 75%DA-CeO2, and 100%DA-CeO2. The nomenclature is based on the proportion of arginine to PAA added during the synthesis. Among them, 100%LA-CeO2and 100%DA-CeO2were found to be insoluble in water, whereas PAA-CeO2, 25° / oLA-CeO2, 50° / oL7\-CeO2, 75%LA-CeO2, 25%DA-CeO2, 50%DA- CeO2, and 75%DA-CeO2exhibited solubility in water and displayed ultrasmall clusters, as confirmed in Figure 2. The XRD patterns depicted in Figure 3a elucidate the composition of ultrasmall clusters as CeO2. Within the XRD spectrum, notable broad shoulder peaks emerge within the 25-30° (20), 45-50° (20), and 55-60° (20) ranges, corresponding to the (111), (220), and (222) crystallographic planes of CeO2. The FTIR analysis presented in Figure 3b further substantiates the modification of CeO2by D-Arg or L-Arg. In consideration of the catalytic activity's dependence on the cerium valence state, XPS analysis was conducted, as illustrated in Figure 4. Upon a more comprehensive introduction of D-Arg or L-Arg, there is an increased abundance of tetravalent cerium, with 75%LA-CeO2and 75%DA-CeO2exhibiting the highest content of

[0166] Ce4+. This observation affirms that arginine plays a pivotal role in regulating the structural characteristics of CeCh.

[0167] Arginine regulates the aoyrase-like activity of CeC

[0168] As Ce4+exhibits higher affinity for ATP, we conducted a detailed investigation into its capacity to scavenge ATP. As depicted in Figure 5, our findings reveal that 75%LA-CeO2 and 75%DA-CeO2 exhibit enhanced ATP scavenging capabilities, with activity positively correlated with the concentration of these materials. Notably, the activity of 75%DA-CeO2 surpasses that of 75%LA-CeO2. Consequently, in subsequent in vivo studies, we have chosen 75%DA-CeO2 for further exploration in the treatment of inflammatory diseases. Furthermore, HPLC analysis was employed to delineate the hydrolysis profile of ATP (Figure 6). The results indicate a discernible reduction in the characteristic peak of ATP substrate and the emergence of new peaks associated with ADP / AMP products after incubation with different CeC samples. This confirms the catalytic activity of CeO2 akin to that of natural apyrase. Notably, among these samples, 75%DA- CeC>2 and 75%LA-CeO2 demonstrated the highest conversion rate. of 75%DA-CeC>2 with other traditional

[0169] Several studies in the literature have illustrated that lanthanide nanoparticles possess the capability to scavenge ATP owing to the robust binding affinity between lanthanide ions and the phosphate group of ATP. In this context, we conducted a comparative assessment of the ATP-scavenging abilities of PCN- 222, PCN-777, ZrSe2, and LF-UCNP in comparison with 75o / oDA-CeO2. The findings unequivocally established that our 75%DA-CeO2 exhibited the highest ATP-scavenging ability among the tested materials (Figure 7). The control in this experiment is the HEPES buffer, as indicated in Figure 5. of 75%DA-CeO2 with natural

[0170] Natural enzymes exhibit high enzymatic activity; therefore, we further tested the activity of natural apyrase arginine (Figure 8). We observed that apyrase could rapidly clear ATP, but natural enzymes were highly unstable. The enzymatic activity in the second round of testing decreased significantly compared to the first, solely due to repeated freeze-thaw cycles. Subsequently, we examined whether the enzymatic activity of natural apyrase underwent changes after exposure to the gastrointestinal (GI) environment. We assessed the enzyme's activity after incubating it in simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and simulated colon fluid (SCF) for a certain duration. The results indicated that natural apyrase could not withstand the challenges of the GI tract; its activity decreased to zero after exposure to SGF and SCF, whereas it remained unaffected by SIF. In summary, our 75%DA-CeO2 exhibited superior catalytic activity compared to traditional nanozymes and demonstrated robust stability relative to natural apyrase.

[0171] Arginine regulates other enzyme-like activity of CeCh

[0172] CeOz, in addition to simulating the activity of the hydrolytic enzyme apyrase, can also mimic redox enzymes such as catalase (CAT), superoxide dismutase (SOD), oxidase (OXD), and peroxidase (POD). Therefore, in our subsequent tests, we explored whether the regulation by arginine would affect the activity of other enzymes-like. The results revealed that this series of CeO? can also simulate the activity of SOD, but this activity is not influenced by the regulation of arginine (Figure 9). Hence, we devised a method utilizing arginine for the specific modulation of apyrase-like activity of CeOz.

[0173] Large-scale production and stability of 75%DA-CeO?

[0174] The system for producing 75%DA-CeO2 can be scaled up from a few milliliters to several liters, making it suitable for large-scale production (Figure 10). Additionally, we conducted tests to evaluate the dispersibility and long-term stability of 75%DA-CeO2 in aqueous solutions. Our findings indicate that 75%DA-CeO2 exhibits excellent water dispersibility and remains stable without any observable sedimentation or changes for at least 30 days (Figure 11).

[0175] Moreover, their capacity for ATP scavenging has not been affected (Figure 12). This comprehensive analysis serves as robust evidence of the water solubility and stability of our synthesized material.

[0176] Antioxidant artificial enzvmes-armed probiotics (CB@75%DA- could be flexibly constructed

[0177] Due to the potent ATP-clearing and R.OS-clearing capabilities of CeCh, as well as its ability to alleviate inflammation, we plan to initially use it for treating inflammatory bowel disease (IBD). However, due to the small size of these nanoparticles, they are easily metabolized and cleared, making it challenging to sustain prolonged anti-inflammatory effects. Therefore, we aim to attach CeCh to probiotics, aiming to increase the retention time of CeC in the intestines and enhance its sustained antioxidant capabilities. To this, after synthesizing the artificial enzyme 75%DA-CeO2, we proceeded to integrate it with probiotics of Clostridium butyricum (CB, a well-established anaerobic probiotic with therapeutic potential for IBD), resulting in the formation of the CB@75%DA- CeOz ensemble. The construction of the probiotics-artificial enzymes system, namely CB@75%DA-CeO2, was a straightforward process involving the simple mixing of 75%DA-CeC>2 with CB, due to the coordination between cerium (Ce) with polysaccharides and the phosphate groups present in the bacterial cell wall. The creation of the probiotics-artificial enzymes system, denoted as CB@75%DA-CeC>2, was a straightforward procedure that entailed the simple mixing of 75%DA-CeC>2 with CB, showcasing the compatibility and effectiveness of this combined approach. HAADF-STEM and TEM images revealed the precise positioning of DA-CeCh on the surface of the bacterial cell wall, without inducing any morphological changes to the bacterial structure (Figure 13).

[0178] Furthermore, the incorporation of antioxidant 75%DA-CeC>2 significantly enhanced the ATP-scavenging activity of CB, demonstrating a notable increase in activity with escalating concentrations (Figure 14). This heightened activity ensures the system's robustness in the face of challenging oxidative microenvironments, emphasizing its potential applications in settings characterized by heightened oxidative stress. Having established the ATP- scavenging prowess of the fortified CB@75%DA-CeO2, we delved into their in vitro antioxidant potential using the HT29 cell line as a model. The 75%DA-CeO2 exhibited a capacity to augment the ATP-scavenging ability of CB, resulting in a proportional increase in ATP scavenging with rising concentrations of CB@75%DA-CeO2(Figure 15).

[0179] Stabili in the GI environment.

[0180] Before reaching the inflamed colon, CB@75%DA-CeO2 faces potential challenges in the harsh GI environment post-oral ingestion, including exposure to gastric acid, bile salts, and inflammation, which may destabilize artificial enzymes and deactivate bacteria. Thus, we conducted assessments of the ATP- scavenging ability and viability of CB and CB@75%DA-CeC>2 after incubation in SGF for 10 or 20 minutes, SIF, and SCF for 1 and 2 hours. Fortunately, the ATP- scavenging ability of CB@75%DA-CeO2 remained consistent in SGF, SIF, and SCF for the indicated time points and was distinctly higher than that of CB (Figure 16), suggesting remarkable stability of CB@75%DA-CeO2. Notably, compared to natural apyrase enzymes, 75%DA-CeO2exhibited exceptionally stable catalytic activity even after exposure to the challenging GI environment, signifying outstanding long-term ATP-scavenging and antioxidant abilities (Figure 9). Moreover, arming with 75%DA-CeO2 did not compromise the survival of CB in SGF, SIF, and SCF (Figure 17). ameliorate TNBS-induced Crohn's disease

[0181] After confirming the ATP-scavenging capabilities of our formulation through in vitro experiments, we proceeded to evaluate its therapeutic effectiveness in a murine model of CD. CD was induced in mice through intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS), known to trigger a T-cell- mediated response against hapten-modified autologous proteins / luminal antigens. By day 9, evident body weight loss and bleeding indicated the successful establishment of CD. Subsequently, 25 six-week-old female C57BL / 6 mice with CD were treated with HEPES buffer, 75%DA-CeO2 (1.65 mg / kg), CB (3.5 x 10sCFU / kg), CB (3.5 x 10sCFU / kg) + 75%DA-CeO2(1.65 mg / kg), or CB@75%DA-CeO2(CB: 3.5 x 108CFU / kg, 75%DA-CeO2: 1.35 mg / kg) for three consecutive days. Simultaneously, a control group of five healthy mice received only normal water. Compared to the control groups, CB@75%DA-CeO2demonstrated a significant reduction in weight loss and a decrease in colon length associated with TNBS-induced CD (Figure 18). Similarly, ATP experiments and enzyme-linked immunosorbent assay analysis confirmed that CB@75%DA-CeO2effectively reduced ATP levels in both the colon and feces, along with proinflammatory factors such as IL-18 and IL-6 in the colon (Figure 19). Collectively, these results underscore the superior efficacy of CB@75%DA- CeO2in colitis therapy, highlighting the synergistic effects of 75%DA-CeO2and CB in the ensemble.

[0182] Comparison with clinical IBP drugs

[0183] In this study, we conducted a direct comparison of the efficacy of CB@75%DA- CeO2with conventional biologicals commonly employed in the treatment of CD, such as monoclonal antibodies anti-tumor necrosis factor a (anti-TNFa) or anti- a4p7 integrin blocking antibodies (Figure 20). When stated, mice were injected intraperitoneally with 100 pg anti-mouse TNFo (BE0244, Bio X CeLL) or antimouse a4p7 (BE0034, Bio X CeLL). CB@75%DA-CeO2demonstrated efficacy against Crohn's disease (CD) comparable to established clinical therapies, resulting in body-weight recovery and the preservation of colon length. The favorable outcomes observed with CB@75%DA-CeO2in TNBS-induced CD highlight its potential as a promising candidate for replacing conventional clinical anti-IBD therapies.

[0184] CB@75%DA-CeO2could distinctly ameliorate DSS-induced ulcerative colitis

[0185] To explore the therapeutic potential of CB@75%DA-CeO2across various applications, we assessed its effectiveness in treating UC, another prevalent form of IBD in humans. Colitis was induced in 25 six-week-old female C57BL / 6 mice by administering 3% (w / v) dextran sulfate sodium (DSS) in their drinking water for four consecutive days. Following colitis induction, mice were orally administered different substances daily for four days, including HEPES buffer, 75%DA-CeO2 (1.65 mg / kg), CB (3.5 x 108CFU / kg), CB (3.5 x 108CFU / kg) + 75%DA-CeO2 (1.65 mg / kg), or CB@75%DA-CeO2 (CB: 3.5 x 108CFU / kg, 75%DA-CeO2: 1.35 mg / kg). Additionally, a control group of 5 healthy mice received normal water only. Compared to the control groups, CB@75%DA-CeO2 demonstrated significant mitigation of weight loss and a reduction in colon length associated with DSS-induced colitis (Figure 21).

[0186] Biosafety of CB(3)75o / oDA-CeO2

[0187] To ensure the safe bioapplications of CB@75%DA-CeC>2, a comprehensive assessment of in vivo toxicology was conducted. In the case of both CD and UC mice after treatment, their biochemical and hematological parameters were meticulously investigated (Figure 22-25). Additionally, for long-term toxicity evaluation, healthy mice were orally administered with or without CB@75%DA- CeO2 for 28 days. Subsequently, their biochemical and hematological parameters, along with body-weight changes, were analyzed (Figure 26-28). The comparison between the CB@75%DA-CeC>2-treated group and the control group revealed no distinct differences, indicating negligible toxicity associated with CB@75%DA-CeO2.

[0188] Monosodium iodoacetate (MIA)-induced osteoarthristis (OA) :

[0189] Healthy male SD rats (200 ± 20g, 8 weeks old) were used in this experiment. After 7 days of adaptation feeding, the rats were anesthetized using a small animal gas anesthesia machine, the hair on their right knee joint was shaved, and they were fixed in a supine position. MIA (2 mg / rat) was injected into the right knee joint cavity to establish the OA rat model.

[0190] DA-CeO2 treat OA:

[0191] Three days after modeling, an equal amount of normal saline, free fluorescent probe Cy5.5 solution (Free group), and Cy5.5 fluorescent-labelled DA-CeO2 nanoparticles (NPs group) were injected into the right knee joint cavity of MIA model rats (7 rats per group). The mechanical paw withdrawal threshold of rats was measured, and the baseline pain data after modelling was recorded as the first week on the day of administration, followed by weekly measurements thereafter.

[0192] Description of mechanical paw withdrawal threshold measurement results:

[0193] The mechanical paw withdrawal threshold of each group was around 20g after modeling, indicating successful MIA modeling. Compared to MIA modeling, there were no significant changes in pain threshold in the free drug group. Compared to the MIA modeling group and the free drug group, the pain threshold in the NPs group significantly increased in the 4th and 5th weeks after administration. These results indicate that DA-CeOz nanoparticle formulations can effectively alleviate joint pain in MIA model rats.

[0194] To further explore the regulatory effects of DA-CeCh-modified probiotics on gut dysbiosis, we performed 16S rRNA sequencing on fecal samples from healthy mice and those with TNBS-induced colitis treated with HEPES buffer, 75%DA- CeOz, CB, CB + 75%DA-CeO2, or CB@75%DA-CeO2. TNBS-induced colitis led to reduced microbiome diversity, as reflected by a lower Shannon entropy index (Figure 31a), and altered community composition, as demonstrated by distinct clustering in non-metric multidimensional scaling (NMDS) plots (Figure 31b). At the family level, TNBS treatment significantly reduced Muribaculaceae and Lachnospiraceae while increasing Enterobacteriaceae, hallmark indicators of dysbiosis in IBD patients. Different treatments modulated bacterial diversity and community composition to varying degrees. Notably, CB@75%DA-CeO2 treatment restored Muribaculaceae and Lachnospiraceae populations while reducing Enterobacteriaceae levels in TNBS-induced colitis mice, highlighting its potent therapeutic effects in modulating gut microbiota and alleviating dysbiosis. (Figure 31c-f).

[0195] Comparison with other metal oxide nanoparticles

[0196] The nanoparticles of the present disclosure is compared against metal oxide nanoparticles disclosed in Cao et. al.; Nature Nanotechnology; 18 (6), 617-627. In this article, the nanoparticle is a pyrolyzed Fe@MOF, and further conjugated with a C18-PEG linker for attachment to probiotics. In contrast, DA-CeO? is easily and uniformly prepared and scaled up at room temperature, and can be directly attached to the surface of probiotics without the need for a linker. Further, the dispersibility of the comparator nanoparticle is not as good as that of 75% DA-CeOz and is difficult to synthesize uniformly in large quantities.

[0197] The CB@75%DA-CeOz nanozymes demonstrated stronger anti-inflammatory effects in both ROS and ATP-scavenging abilities (Figure 9a, 9b and 5a). The CB@75%DA-CeOz nanoezymes also demonstrated improved stability with better acid tolerance (Figure 17), and more effective therapeutic effects towards DSS-induced UC (Figure 21a and 21b) and TNBS induced CD (Figure 18a and 18b), where the reduction in weight loss and decrease in clone length nearly reaches the levels of the healthy group when treating CD.

[0198] Conclusion

[0199] In conclusion, we have successfully developed ultrasmall antioxidant CeOz nanozymes featuring water-solubility, scalable production, and long-term stability. Significantly, for the first time, we employed amino acids such as D- Arg to selectively regulate apyrase-like activity without affecting SOD-like activity. These nanozymes showcase remarkable ATP-clearance capabilities and ROS-scavenging ability, underscoring their potential for diverse antiinflammatory applications. When coupled with probiotics, these artificial enzymes can persist in the colon for an extended duration, effectively scavenging ATP, resiliently attenuating inflammation, and serving as a potential treatment for IBD.

[0200] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0201] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0202] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0203] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

Claims1. A metal oxide nanoparticle comprising: a) a metal oxide, the metal capable of being a Lewis acid; b) a polymer comprising an ionisable moiety; and c) an amino acid; wherein a weight ratio of amino acid to polymer is about 0.1 :99.9 to about 99.9:0.1; and wherein the amino acid is electrostatically interacting with the polymer; wherein the metal oxide comprises metal ions in a 3+ and 4+ valence state; and wherein the metal oxide is selected from lanthanide oxide and / or zirconium oxide.

2. The metal oxide according to claim 1, wherein the lanthanide oxide comprises a lanthanide, the lanthanide selected from La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, or a combination thereof.

3. The metal oxide according to claim 1 or 2, wherein the metal oxide is characterised by a 4+ to 3+ valence ratio of about 0.2 to about 0.7.

4. The metal oxide according to any one of claims 1 to 3, wherein the metal ion with a 4+ valence is characterised by a mass percentage about 15% to about 30% relative to the metal oxide nanoparticle.

5. The metal oxide according to any one of claims 1 to 4, wherein the amino acid is a L amino acid, D amino acid or a combination thereof.

6. The metal oxide according to any one of claims 1 to 5, wherein the amino acid is selected from D-arginine (D-Arg), L-arginine (L-Arg), D-methionine CD- Met), L-methionine (L-Met), D-tyrosine (D-Tyr), L-tyrosine (L-Tyr), or a combination thereof.

7. The metal oxide according to any one of claims 1 to 6, wherein the polymer is selected from polyacrylic acid (PAA), Dextra, Guar gum, Gelatin, Xanthan gum, Carboxymethyl cellulose sodium, Carboxymethyl starch (sodium), Crosslinked polyacrylate / C10-30 alkyl acrylate copolymer, Acrylate / C16-20 alkyl ethoxylate (20) cocopolymer, Acrylate / C16-20 alkyl ethoxylate (20) methacrylate copolymer, Acrylate / C18 alkyl ethoxylate (20) methacrylate copolymer, Acrylate / VA copolymer, Sodium acrylate / ethylene / acrylic acid copolymer, Carbomer, and their derivatives thereof.

8. The metal oxide according to any one of claims 1 to 7, wherein the polymer is a polyelectrolyte.

9. The metal oxide according to any one of claims 1 to 8, wherein the polymer is characterised by a molecular weight of about 500 to about 5000.

10. The metal oxide according to any one of claims 1 to 9, wherein the amino acid is configured to interact with metal oxide through coordination to the metal ion via its amino moiety.

11. The metal oxide according to any one of claims 1 to 10, wherein the polymer is configured to interact with metal oxide through coordination to the metal ion via its ionisable moiety.

12. The metal oxide according to any one of claims 1 to 11, wherein the weight ratio of amino acid to polymer is about 75:25.

13. The metal oxide according to any one of claims 1 to 12, wherein a mole ratio of metal oxide to (amino acid and polymer) is about 1: 1 to about 1: 10.

14. The metal oxide according to any one of claims 1 to 13, wherein the metaloxide nanoparticle comprises a corona, the corona comprising a protein, a surfactant, a ligand, or a combination thereof.

15. The metal oxide according to any one of claims 1 to 14, wherein the metal oxide nanoparticle is characterised by an aqueous dispersibility for at least 30 days.

16. The metal oxide according to any one of claims 1 to 15, wherein the metal oxide nanoparticle is attached to a probiotic.

17. An enzyme-probiotic conjugate, comprising the metal oxide nanoparticle according to any one of claims 1 to 16 conjugated to a probiotic.

18. The enzyme-probiotic conjugate according to claim 17, wherein the probiotic is selected from Clostridium butyricum, Bifidobacterium, Lactobacillus, Komagataeibacter, Leuconostoc, or a combination thereof.

19. The enzyme-probiotic conjugate according to claim 17 or 18, wherein the number ratio of enzyme to probiotic is about 1 to about 5000.

20. A use of the metal oxide nanoparticle according to any one of claims 1 to 16 or the enzyme-probiotic conjugate according to any one of claims 17 to 19 as an apyrase mimic and / or a superoxide dismutase mimic.

21. A method of fabricating a metal oxide nanoparticle according to any one of claims 1 to 16, comprising mixing a metal salt, polyelectrolyte and amino acid with a base at ambient temperature.

22. A method of treating an inflammatory disease and / or a wound in a subject in need thereof, comprising administering a therapeutic amount of the metal oxide nanoparticle according to any one of claims 1 to 16 or the enzymeprobiotic conjugate according to any one of claims 17 to 19 to the subject.

23. A metal oxide nanoparticle according to any one of claims 1 to 16 or the enzyme-probiotic conjugate according to any one of claims 17 to 19 for use in treating an inflammatory disease and / or a wound.

24. Use of a metal oxide nanoparticle according to any one of claims 1 to 16 or the enzyme-probiotic conjugate according to any one of claims 17 to 19 in the manufacture of a medicament for the treatment of an inflammatory disease and / or a wound.

25. The method, metal oxide nanoparticle or the enzyme-probiotic conjugate for use or use according to any one of claims 22 to 24, wherein the inflammatory disease is characterised by an elevated extracellular ATP release relative to a control.

26. The method, metal oxide nanoparticle or the enzyme-probiotic conjugate for use or use according to any one of claims 22 to 25, wherein the inflammatory disease is selected from inflammatory bowel disease (IBD), osteoarthritis, stroke, acute kidney injury, acute liver injury, myocardial infarction, colitis, ulcerative colitis, and Crohn's disease.

Images