Antimicrobial composite materials

Abstract

The invention provides an antimicrobial composite material, comprising a bacterial-enzyme- degradable anionic copolymer, and a plurality of metal ions, wherein the bacterial-enzyme- degradable anionic copolymer and the plurality of metal ions form a complex. The invention also provides a pharmaceutical composition and a composition comprising the antimicrobial composite material. In addition, the invention provides the antimicrobial composite material and the pharmaceutical composition for use as a medicament.

Description

[0001] ANTIMICROBIAL COMPOSITE MATERIALS

[0002] FIELD OF INVENTION

[0003] The present invention provides antimicrobial composite materials, more particularly, antimicrobial composite materials suitable for delivery of metal ions.

[0004] BACKGROUND

[0005] The listing or discussion of a poor-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] Metal ions have emerged as pivotal agents in modern medicine, offering unique therapeutic avenues across a spectrum of diseases, including cancer, infections, metabolic disorders, and cardiovascular conditions. Their distinct physicochemical properties — such as redox activity, coordination versatility, and catalytic potential — enable interactions with biological targets that are often inaccessible to traditional organic compounds. For instance, platinum-based complexes like cisplatin have revolutionized oncology by inducing DNA damage in tumour cells, while ions such as silver and gallium exhibit antimicrobial properties and modulate immune responses. Despite the proven efficacy, metal ions lack selectivity, thus raising concerns about their biocompatibility. Consider the case where free silver ions could induce oxidative stress to organelles, causing structural and functional damage that may result in cellular injury and systemic toxicity. Moreover, free ions are often rapidly cleared from the body, reducing their effectiveness. Mechanisms such as ion exchange, electrostatic interactions, and microprecipitation facilitate their binding and metabolism by cells. And they can be captured and cleared by chelators in the blood, such as transferrin, which, while primarily responsible for iron transport, can also bind other metal ions, leading to their removal from circulation.

[0007] In the context of infectious diseases, metal ions have a long history of use in infection control and have gained more interest in recent years in light of the emerging crisis of antimicrobial resistance (AMR). For example, despite its extensive history and effectiveness against bacteria, the exact bactericidal mechanism of silver has remained unclear until recently. It was found that Ag+could increase membrane permeability and induce reactive oxygen species (ROS) production in Gram-negative bacteria, which could also enhance the performance of other antibiotics. Iron has also emerged as a promising antimicrobial agent, particularly following the recent discovery of Fe-mediated ferroptosis-like bacterial cell death. However, their clinical application necessitates careful consideration of delivery strategies to maximize efficacy while minimizing potential side effects. A primary concern of the delivery system is the off-target toxicity associated with free metal ions. To address this, encapsulation techniques — such as complexation or coordination — should be employed to sequester metal ions within carriers, preventing premature interactions with healthy tissues. Targeted release at infection sites is another critical factor, as the delivery system must facilitate the release of metal ions to exert their antimicrobial effects once at the infection site. This requires the system to be responsive to specific infection-associated microenvironmental triggers — such as abnormal pH changes, enzymatic activity, or redox conditions. Moreover, it's imperative that these delivery systems do not compromise the intrinsic antimicrobial activity of the metal ions. On the contrary, they should enhance efficacy, potentially allowing for reduced dosages.

[0008] Thus, there is a need for alternative and / or improved antimicrobial composite material suitable for the delivery of metal ions that can overcome the above-mentioned problems.

[0009] SUMMARY

[0010] Aspects and embodiments of the current invention will now be described by reference to the following numbered clauses.

[0011] 1 . An antimicrobial composite material, comprising: a bacterial-enzyme-degradable anionic copolymer; and a plurality of metal ions, wherein the bacterial-enzyme-degradable anionic copolymer and the plurality of metal ions form a complex.

[0012] 2. The antimicrobial composite material according to Clause 1 , wherein the plurality of metal ions comprise metal ions that can form two or more (e.g. 2, 3, 4, 5 or 6) ionic bonds with the bacterial-enzyme-degradable anionic copolymer.

[0013] 3. The antimicrobial composite material according to Clause 1 or Clause 2, wherein the plurality of metal ions are selected from one or more of the group consisting of Ag+, Fe2+, Fe3+, Cu2+, Ni2+, Mn2+, Co2+, Zn2+, Ga3+, Ca2+, Mg2+, lr3+, Ce3+, and Ce4+.

[0014] 4. The antimicrobial composite material according to any one of the preceding clauses, wherein the plurality of metal ions comprise Ag+and Fe2+, optionally wherein the molar ratio of Ag+to Fe2+is from 0.01 :1 to 5:1 , such as from 0.02:1 to 2:1 , such as from 0.1 :1 to 1 :1 , such as about 0.2:1.

[0015] 5. The antimicrobial composite material according to Clause 4, wherein the complex formed between the bacterial-enzyme-degradable anionic copolymer and the plurality of metal ions provides nanostructures.

[0016] 6. The antimicrobial composite material according to any one of the preceding clauses, wherein the antimicrobial composite material is in the form of particles, having a mean hydrodynamic diameter of from 100 to 500 nm, such as from 125 to 300 nm, such as from 155 to 291 nm, such as from 166 to 208 nm, such as about 175 nm.

[0017] 7. The antimicrobial composite material according to any one of the preceding clauses, wherein the molar ratio of the bacterial-enzyme-degradable anionic copolymer to the collective total of metal ions is from 1 :1 to 5:1 , such as from 1 :0.67 to 4:1 , such as about 3.33:1 .

[0018] 8. The antimicrobial composite material according to any one of the preceding clauses, wherein the bacterial-enzyme-degradable anionic copolymer has the formula (I): where:

[0019] R is a Ci to C linear or branched alkylene chain;

[0020] A represents C=O or P(ORa)=O;

[0021] Rarepresents a linear or branched Ci to Ce alkyl group; a represents a block polyethylene glycol unit having a number average molecular weight of from 500 to 10,000 Daltons; b and c together represent a random copolymer block, where b and c each independently have a value of from 8 to 100.

[0022] 9. The antimicrobial composite material according to Clause 8, wherein one or more of the following apply:

[0023] (a) a represents a block polyethylene glycol unit having a number average molecular weight of about 2,000 Daltons;

[0024] (b) b and c each independently have a value of from 10 to 50, such as 15 to 20, such as 18;

[0025] (c) A represents C=O; and

[0026] (d) R represents -(CH2)5-.

[0027] 10. The antimicrobial composite material according to Clause 8 or Clause 9, wherein the bacterial-enzyme-degradable anionic copolymer has a number average molecular weight of from 6,000 to 10,000 Daltons, such as from 6,900 to 8,000 Daltons.

[0028] 11 . The antimicrobial composite material according to any one of the preceding clauses, wherein the bacterial-enzyme-degradable anionic copolymer is degradable by a bacterial- secreted lipase or a bacterial-secreted phosphoesterase, such as a bacterial-secreted lipase.

[0029] 12. The antimicrobial composite material according to any one of the preceding clauses, wherein the antimicrobial composite material has a zeta potential of from -10 to -60 mV, such as from -34 to -52.1 mV, such as from -36.4 to -49.6 mV, such as about -39.8 mV.

[0030] 13. A pharmaceutical composition comprising an antimicrobial composite material according to any one of Clauses 1 to 12 and one or more of a pharmaceutically acceptable excipient and carrier.

[0031] 14. An antimicrobial composite material according to any one of Clauses 1 to 12, or a pharmaceutical composition as described in Clause 13, for use as a medicament.

[0032] 15. A method of treating a subject suffering from a microbial and / or fungal infection comprising the steps of administering to the subject a therapeutically effective amount of an antimicrobial composite material according to any one of Clauses 1 to 12, or a pharmaceutical composition as described in Clause 13, such that the infection is treated. 16. Use of an antimicrobial composite material according to any one of Clauses 1 to 12, or a pharmaceutical composition as described in Clause 13, in the manufacture of a medicament to treat a microbial and / or fungal infection in a subject in need thereof.

[0033] 17. An antimicrobial composite material according to any one of Clauses 1 to 12, or a pharmaceutical composition as described in Clause 13, for use in the treatment of a microbial and / or fungal infection.

[0034] 18. A composition comprising an antimicrobial composite material according to any one of Clauses 1 to 12, optionally wherein the composition is a cosmetic composition or a detergent composition.

[0035] BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1 is a schematic illustration of the (A) fabrication of the Fe@Ag complex and (B) its proposed mechanism of action.

[0037] FIG. 2 depicts (A) Checkerboard assay of free Ag+and Fe2+against P. aeruginosa. (B) Bactericidal activity of different complexes at various concentrations. The concentrations are presented by folds of MIC. (C) The inhibition curves of the Complex 3 compared to free iion combination (ions alone). (D) Evolution of antibiotic resistance in P.aeruginosa, in response to the treatment of complex or the combination of free Ag+and Fe2+ions. Data are presented as the highest concentration that allowed for visible bacterial growth and presented as the fold of MIC of the first generation (n=3).

[0038] FIG. 3 depicts the in vitro ion release and bacteria-killing study. (A) In vitro release profile of Fe and (B) silver in P.aeruginosa , E.faecalis supernatant, and blank culture media. (C) The time-killing curve of the complex, (D) the simple combination of free Ag+and Fe2+ions, (E) free Fe2+ions, and (F) free Ag+ion. The concentrations in all four panels are presented as folds of MIC of the complex.

[0039] FIG. 4 depicts the mechanism study of the antibacterial activity of the complex. (A) PI fluorescence intensity for membrane damage study (B) The fluorescence intensity of reduced and (C) oxidated forms of BODIPY-C11 after sublethal treatment of different agents. (D) The fluorescence intensity of DCFH-DA for ROS generation study. (E) Volcano plot of DEGs (Red: upregulated genes; blue: downregulated genes, gray: no significance). (F) Top 20 GO enrichment analysis by number of genes. (G) Top 10 KEGG enrichment of DEGs by number of genes.

[0040] FIG. 5 depicts the in vitro and in vivo toxicity of the Fe@Ag complex. (A) In vitro cell viability assay of the complex and free ions. (B) Body weight change of healthy mice after different treatments (n=5). (C-E) Concentrations of metals in lungs and livers at 3- and 24-h postadministration (n=4). (G) H&E staining of lungs and livers under different treatments (scale bar 100 pm). Pathological changes of lung tissue included lung edema, alveolar wall thickening, and larger alveolar space.

[0041] FIG. 6 depicts the in vivo therapeutic efficacy against acute bacterial pneumonia model. (A-B) Bacterial loads of lungs and livers after different treatments (n=5). (C) Bodyweight change and (D) survival status of infected mice at lethal bacterial dosage (n=5).

[0042] FIG. 7 depicts the characterization of the Ag@Fe complex. (A) Size value and (B) size graph of complex 1 -5. (C) Zeta potential of copmlexl -5. (D) TEM image of complex 3.

[0043] FIG. 8 depicts the characterization of the Fe Complex and Ag Complex. (A) and (B) hydrodynamic size value of Fe complex and Ag complex, respectively. (C) and (D) Zeta potential of Fe Complex and Ag Complex, respectively.

[0044] FIG. 9 depicts the cytotoxicity of (A) Fe2+, (B) Ag+, (C) Fe Complex, and (D) Ag Complex.

[0045] DESCRIPTION

[0046] The present inventors have developed a copolymer-based antimicrobial composite material (including PEG-b-(PCL-co-PPA)) for the delivery of metal ions. The copolymer comprises potent chelating agents (such as phosphonic acid groups), which enable strong bindings to encapsulate the metal ions by complexation through multiple electron pair donors. Additionally, the copolymer exhibits degradability to specific stimulus (i.e., presence of bacterial enzyme, such as bacterial lipase) making it an ideal responsive material to infection signals for the specific release of cargo. As demonstrated in the Examples of the present disclosure, the antimicrobial composite material exhibits effective in vitro and in vivo antimicrobial performance, particularly against P.aeruginosa-induced pneumonia, without apparent toxicity, underscoring its potential for translational application in the field of metal ion drug delivery. Thus, in a first aspect of the invention, there is provided an antimicrobial composite material, comprising: a bacterial-enzyme-degradable anionic copolymer; and a plurality of metal ions, wherein the bacterial-enzyme-degradable anionic copolymer and the plurality of metal ions form a complex.

[0047] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

[0048] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0049] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of two or more such compounds, reference to “a composition” includes mixtures of two or more such compositions, and the like.

[0050] As used herein, the term “bacterial-enzyme-degradable anionic copolymer” refers to a polymer consisting of at least two types of constituent units with overall negative charge attributed to the presence of negatively charged groups attached to the polymer backbone. These negative charged groups interact with the plurality of metal ions to form complexes. Further, the polymer comprises one or more functional groups that can be broken down by a suitable bacterial enzyme (i.e., biodegradable functional groups).

[0051] Any suitable biodegradable functional group may be used herein. When used herein, the term biodegradable functional group is intended to refer to a functional group that can be cleaved in the environment and / or in vivo either by biological materials present in the ambient environment in which an oligomer, polymer or molecule of the current invention may find itself in. Non-limiting examples of biodegradable functional groups that may be mentioned herein include urea, carbamate, acetal, amide, ester, carbonate ester, urethane, disulfide, anhydride, and hydrazone. Said functional groups may be susceptible to cleavage by biological materials in the ambient environment (e.g. esters may be cleaved due to the presence of enzymes). This cleavage may take place in vivo or ex vivo, depending on the way that the materials disclosed herein are used and / or disposed of. Examples of functional groups that may not be biodegradable include ether linkages.

[0052] As mentioned above, the bacterial-enzyme-degradable anionic copolymer and the plurality of metal ions according to the present disclosure form a complex. Each of the plurality of metal ions can form interactions via ionic bonds and / or coordination bonds with two or more donor atoms present in the bacterial-enzyme-degradable anionic copolymer (such as deprotonated oxygen atoms in phosphonic acid groups). As such, in certain embodiments, the plurality of metal ions may comprise metal ions that can form two or more (e.g. 2, 3, 4, 5 or 6) ionic bonds with the bacterial-enzyme-degradable anionic copolymer.

[0053] The antimicrobial composite material according to the present disclosure is a useful delivery platform for releasing metal ions for various applications, including therapeutics. As such, in certain embodiments, the plurality of metal ions may be selected from one or more of the group consisting of Ag+, Fe2+, Fe3+, Cu2+, Ni2+, Mn2+, Co2+, Zn2+, Ga3+, Ca2+, Mg2+, lr3+, Ce3+, and Ce4+.

[0054] In certain embodiments of the invention that may be mentioned herein, Ag+may be present. As such, the plurality of metal ions may be Ag+and one or more of the group consisting of Fe2+, Fe3+, Cu2+, Ni2+, Mn2+, Co2+, Zn2+, Ga3+, Ca2+, Mg2+, lr3+, Ce3+, and Ce4+. In other embodiments of the invention that may be mentioned herein, Ag+may be absent. As such, the plurality of metal ions may be one or more of the group consisting of Fe2+, Fe3+, Cu2+, Ni2+, Mn2+, Co2+, Zn2+, Ga3+, Ca2+, Mg2+, lr3+, Ce3+, and Ce4+.

[0055] As demonstrated in the Examples of the present disclosure, the combination of Fe2+and Ag+exhibits synergistic effects and enhanced antibacterial activity against pathogens such as P. aeruginosa. As such, in certain embodiments, the plurality of metal ions may comprise Ag+and Fe2+. In more particular embodiments, the molar ratio of Ag+to Fe2+may be from 0.01 :1 to 5:1 , such as from 0.02:1 to 2:1 , such as from 0.1 :1 to 1 :1 , such as about 0.2:1 . The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, within 1%, within 0.5%, within 0.1 %, within 0.05%, within 0.01 %, within 0.005%, or within 0.001% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0056] For the avoidance of doubt, the antimicrobial composite material according to the present disclosure exclude salts of the anionic copolymer. As mentioned above, the bacterial-enzyme- degradable anionic copolymer and the plurality of metal ions form a complex. In certain embodiments, the complex formed between the bacterial-enzyme-degradable anionic copolymer and the plurality of metal ions may provide nanostructures, such as nanoparticles and the like.

[0057] In certain embodiments, the antimicrobial composite material may be in the form of particles having a mean hydrodynamic diameter of from 100 to 500 nm, such as from 125 to 300 nm, such as from 155 to 291 nm, such as from 166 to 208 nm, such as about 175 nm. The mean hydrodynamic diameter may be measured by conventional techniques (such as dynamic light scattering (DLS)), and / or devices (such as the Malvern Nano-ZS Zetasizer) known in the art.

[0058] The bacterial-enzyme-degradable anionic copolymer and the plurality of metal ions may be present in any suitable amount. In certain embodiments, the molar ratio of the bacterial- enzyme-degradable anionic copolymer to the collective total of metal ions may be from 1 :1 to 5:1 , such as from 1 :0.67 to 4:1 , such as about 3.33:1 .

[0059] In certain embodiments, the bacterial-enzyme-degradable anionic copolymer may have the formula (I):

[0060] where:

[0061] R is a Ci to C10 linear or branched alkylene chain;

[0062] A represents C=O or P(ORa)=O;

[0063] Rarepresents a linear or branched Ci to Cg alkyl group; a represents a block polyethylene glycol unit having a number average molecular weight of from 500 to 10,000 Daltons; b and c together represent a random copolymer block, where b and c each independently have a value of from 8 to 100.

[0064] Unless otherwise stated, the term “alkyl” refers to an unbranched or branched, cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl) hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms). Where the term “alkyl” refers to an acyclic group, it is preferably C1-10 alkyl and, more preferably, C1-6 alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl). Where the term “alkyl” is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C3-12 cycloalkyl and, more preferably, Cs-io (e.g. C5-7) cycloalkyl. In certain preferred embodiments of the invention, Rarepresents a linear or branched Ci to Cg alkyl group.

[0065] The term alkylene may be interpreted accordingly. As will be appreciated, the term “alkylene” is intended to refer to a hydrocarbyl radical that sits between two other groups (and so is not chain terminating) and may, for example, be a hydrocarbyl radical of the type X-CRxRy-X’, where X and X’ are the groups to which the alkyene radical is attached and Rxand Rymay be any suitable group, such as an alkyl, halo or together with the carbon atom to which they are attached form a cycloalkyl ring. In certain preferred embodiments of the invention, R is a Ci to C10 linear or branched alkylene chain.

[0066] In certain embodiments, one or more of the following may apply:

[0067] (a) a represents a block polyethylene glycol unit having a number average molecular weight of about 2,000 Daltons;

[0068] (b) b and c each independently have a value of from 10 to 50, such as 15 to 20, such as 18;

[0069] (c) A represents C=O; and

[0070] (d) R represents -(CH2)5-

[0071] In certain embodiments, the bacterial-enzyme-degradable anionic copolymer may have a number average molecular weight of from 6,000 to 10,000 Daltons, such as from 6,900 to 8,000 Daltons. The molecular weight may be determined by conventional techniques (such as gel permeation chromatography) and / or devices (such as the Agilent 1260 Infinity GPC / SEC System using the PLgel MIXED-C, 5 pm, column and DMF as the solvent) known in the art.

[0072] As mentioned above, the bacterial-enzyme-degradable anionic copolymer according to the present disclosure can be broken down in the presence of a suitable bacterial enzyme that is capable of cleaving the biodegradable functional groups in the copolymer. In certain embodiments, the bacterial-enzyme-degradable anionic copolymer may be degradable by a bacterial-secreted lipase or a bacterial-secreted phosphoesterase, such as a bacterial- secreted lipase.

[0073] In certain embodiments, the antimicrobial composite material may have a zeta potential of from -10 to -60 mV, such as from -34 to -52.1 mV, such as from -36.4 to -49.6 mV, such as about -39.8 mV. The zeta potential may be measured by conventional techniques (such as dynamic light scattering (DLS)), and / or devices, (such as the Malvern Nano-ZS Zetasizer) known in the art.

[0074] The antibacterial composite material disclosed herein may be used in a pharmaceutical composition. Thus, in second aspect of the invention, there is provided a pharmaceutical composition comprising an antimicrobial composite material as described hereinbefore and one or more of a pharmaceutically acceptable excipient and carrier.

[0075] Antimicrobial composite materials according to the current invention may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.

[0076] Antimicrobial composite materials according to the current invention will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

[0077] Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice.

[0078] The amount of the antimicrobial composite material according to the current invention in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is / are employed. In any event, the amount of the antimicrobial composite materials according to the current invention in the formulation may be determined routinely by the skilled person.

[0079] For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w / w) active ingredient (i.e. the antimicrobial composite material according to the current invention); from 0 to 99% (w / w) diluent or filler; from 0 to 20% (w / w) of a disintegrant; from 0 to 5% (w / w) of a lubricant; from 0 to 5% (w / w) of a flow aid; from 0 to 50% (w / w) of a granulating agent or binder; from 0 to 5% (w / w) of an antioxidant; and from 0 to 5% (w / w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w / w) of a release-controlling polymer.

[0080] A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w / w) active ingredient; and from 50% (w / w) to 99% (w / w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w / w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

[0081] Depending on the disorder, and the patient, to be treated, as well as the route of administration, the antimicrobial composite materials according to the current invention may be administered at varying therapeutically effective doses to a patient in need thereof.

[0082] As will be appreciated, the antimicrobial composite material and its pharmaceutical composition (formulations) may be used in medicine. Thus, in a further aspect of the invention, there is provided an antimicrobial composite material or a pharmaceutical composition as described hereinbefore for use as a medicament.

[0083] In a further aspect of the invention, there is provided a method of treating a subject suffering from a microbial and / or fungal infection comprising the steps of administering to the subject a therapeutically effective amount of an antimicrobial composite material or a pharmaceutical composition as described hereinbefore, such that the infection is treated.

[0084] In a further aspect of the invention, there is provided a use of an antimicrobial composite material or a pharmaceutical composition as described hereinbefore, in the manufacture of a medicament to treat a microbial and / or fungal infection in a subject in need thereof.

[0085] In a further aspect of the invention, there is provided a use of an antimicrobial composite material or a pharmaceutical composition as described hereinbefore, for use in the treatment of a microbial and / or fungal infection.

[0086] For the avoidance of doubt, in the context of the present invention, the term “treatment’ includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and / or diagnosis of patients which are susceptible to the relevant disease states. The terms “patient” and “patients” include references to mammalian (e g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

[0087] The term “effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

[0088] However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage / severity of the disease.

[0089] Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of an antimicrobial composite material according to the current invention.

[0090] In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0091] As will be appreciated, the antimicrobial composite material may be used in various applications and / or compositions, including a cosmetic composition or a detergent composition. Thus, in a further aspect of the invention, there is provided a composition comprising an antimicrobial composite material as described hereinbefore. In certain embodiments, the composition may be a cosmetic composition or a detergent composition.

[0092] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.

[0093] EXAMPLES

[0094] In the present Examples, a block copolymer consisting of polyethylene glycol) (PEG) and an anionic and lipase degradable block of polyphosphonic acid (PPA)- and polycaprolactone (PCL)-based copolymer, PEG-b-(PCL-co-PPA) (FIG. 1A), for the delivery of antimicrobial metal ions was designed. Recognized as potent chelating agents, phosphonic acids contain a tetrahedral phosphorus atom in a +5 oxidation state, which, through multiple sigma-electron pair donors and an oxygen-based coordination platform, enables strong bindings to encapsulate the metal ions by complexation. Additionally, PCL exhibits degradability to bacterial lipase, making it an ideal responsive material to infection signals for the specific release of cargo. The complex formed by the PEG-b-(PCL-co-PPA) copolymer and metal ions with inherent antimicrobial properties, specifically Fe2+and Ag+(FIG. 1) was investigated, and its antibacterial efficacy evaluated. P. aeruginosa was selected as the model organism due to its clinical relevance and role as a main cause of hospital-acquired pneumonia. As a Gramnegative bacterium, its outer membrane limits antibiotic penetration, contributing to intrinsic resistance. Together with its remarkable ability to acquire additional resistance mechanisms, these features make it a key member of the ESKAPE pathogens, a group of bacteria that are major contributors to antimicrobial resistance and are known for their ability to "escape" the effects of existing antibiotics. The present findings demonstrated that the complex selectively released free metal ions in response to the P.aeruginosa microenvironment, and the combination of Fez+and Ag+exhibited synergistic effects and enhanced antibacterial activity. Furthermore, the biosafety of the complex was validated through both in vitro and in vivo assessments, and its therapeutic efficacy was confirmed using an in vivo acute bacterial pneumonia model.

[0095] Materials and methods

[0096] BODIPY-C1 1 was purchased from Thermo Fisher. Total RNA Isolation Kit V2-RC112 was purchased from Vazyme LLC. Cell Counting Kit-8 (CCK-8) was purchased from GIpBio. Amicon® Ultra-4 Centrifugal Filter Units (100k MWCO) purchased from Merck. All other chemicals and materials were purchased from Sigma-Aldrich unless stated otherwise. The PEG-b-(PCL-co-PPA) was synthesized according to the published method (B. Zhang, D. Lu, D.B.R. Wang, Z.Y. Kok, M.B. Chan-Park, H. Duan, Enzyme-Responsive Polyion Complex Nanoparticles of Cationic Antimicrobials for Activatable Antibacterial Therapy, Advanced Functional Materials 34(46) (2024) 2407869). P.aeruginosa PAO1 and NIH / 3T3 cell lines were obtained from ATCC and used according to the protocols. BALB / c mice were purchased from InVivos Pte Ltd. and used according to protocols.

[0097] The size and zeta potential of the nanoparticles were measured by the Malvern Nano-ZS Zetasizer. Transmission electron microscopy (TEM) images were captured by a JEM-1400 Flash Electron Microscope. The optical density of bacterial suspension was measured by a Perkin Elmer EnSpire Multimode Plate Reader. Flow cytometric information was obtained by a BD 5-Lasers Fortessa X20 equipment. The metal ion concentrations were measured by an Agilent 5800 inductively coupled plasma-optical emission spectrometry (ICP-OES) spectrometer. Tissue frozen section was prepared by a Leica CryoStat CM1950 freezing microtome. RNA-seq was conducted by Azenta Life Sciences Inc.

[0098] Example 1 : Preparation of the metal ion-copolymer complex

[0099] FeSO4'7H2O (Fe2+concentration is 1 .25 mM) solution was added to 2.1 mg / mL copolymer (molar concentration of phosphonic acid (PA) is 5 mM) dropwise under stirring, then AgNOs (Ag+concentration is 1 .25 mM) solution was added to the mixture dropwise. Different compositions of complex were obtained by changing the mixing ratio. After 24 hours of stirring, the unreacted polymer and free ions were removed by ultrafiltration (100k MWCO).

[0100] Results and Discussion:

[0101] The metal ion-copolymer complex was prepared by mixing Fe2+with the copolymer solution, followed by adding Ag+solutions. Different ratios of mixing were explored and summarized in Table 1. With the Ag+component increasing, an increasing trend in the zeta potential of the complex accompanied by an increase in their hydrodynamic size was observed (Table 1 and FIG. 7). And turbidity was observed once the phosphonic acid (PA)-Fe-Ag ratio reached 4:1 :1 , as represented by Complex 2. This is possibly resulting from the reduction of colloidal stability as this ratio exceeded the maximum chelating capability of the copolymer. Therefore, Complex 3 (FIG. 7D) was selected for further studies as this ratio allows maximum Ag content that no precipitates were formed during preparation.

[0102] Table 1. and characterization of the

[0103] >

[0104] Complex PA:Fe:Ag Stability Size (nm) Zeta (mV) Molar ratio Intensity mean

[0105] 1 ’ 4:1 :2 ’ Turbid ‘ 291 ‘ -34.3

[0106] 2 4:1 :1 Turbid 208 -36.4

[0107] 3 4-1 ■- Homogenous 175 -39.8

[0108] ■5

[0109] 4 4-1 ■— Homogenous 166 -49.6

[0110] ‘10

[0111] 5 4-1 ■— Homogenous 155 -52.1

[0112] ‘50

[0113] Example 2: In vitro antibacterial study

[0114] A colony of P.aeruginosa PAO1 was inoculated in Luria-Bertani (LB) Broth at 37 °C overnight with shaking at 280 rpm. 10 pL of bacterial suspension from overnight culture was diluted 100 times with fresh broth and incubated in 37 °C for another 4 h to reach mid-exponential phase.

[0115] Then the P.aeruginosa were diluted by Mueller Hinton Broth (MHB) to obtain a CFUs~107per mL, before treated with different drugs. The plates were placed in a 37 °C incubator with and shaking at 280 rpm for 18 h. Then OD6oo was measured by a microplate reader. The growth inhibition rate was calculated as below. MIC value was the lowest concentration that inhibited

[0116] 90% of growth.

[0117] Growth Inhibition (

[0118] Where ODXis the OD6ooof each well, ODctriis the OD50oof untreated bacterial culture (negative control), and blank is the ODeooof nutrient solution only.

[0119] For determination of MBC, 10 pL of the culture after mixing with drugs for 18 h was withdrawn and the bacteria were counted by a Standard Plate Count method. Briefly, 10 pL of the culture was taken and 10x serially diluted (10 pL concentrated mixture was added to 90 pL PBS to reach a 10xdilution) for 6 times (highest dilution was 106x dilution). 5 pL of each dilution was transferred to a LB agar plate and incubated at 37 °C. After 24 h, the lowest dilution with countable bacteria CFUs was selected for counting.

[0120] For FIC calculation: FIC MIC Ag in combination MIC Fe in combination MIC Ag alone MIC Fe alone

[0121] For investigating the time-killing efficacy of the drugs, 100 pL of 1 x 107CFU / mL log phase P.aeruginosa was treated with 100 pL of different concentrations of drugs in MHB at 37 °C with shaking at 280 rpm for 0.5, 1 , 3, 6, and 24 h. And the bacteria were counted by a Standard Plate Count method as mentioned.

[0122] Results and Discussion:

[0123] Prior to testing the complex, the synergism of Ag+and Fe+ions was first verified by a checkerboard assay (FIG. 2A), which was indicated by the fractional inhibitory concentration (FIC) of 0.14. A synergistic effect can be concluded if the FIC index is less than 0.5, and this result was also in line with the previous report of an antibacterial synergistic combination by Ag-Fe bimetallic nanoparticle (A.L. Padilla-Cruz, J.A. Garza-Cervantes, X.G. Vasto-Anzaldo, G. Garcia-Rivas, A. Leon-Buitimea, J.R. Morones-Ramirez, Synthesis and design of Ag-Fe bimetallic nanoparticles as antimicrobial synergistic combination therapies against clinically relevant pathogens, Scientific Reports 1 1 (1) (2021 ) 5351 ). Then the in vitro antibacterial efficacy of the complexes was evaluated. As shown in Table 2, all 5 complexes had a singledigit minimum inhibitory concentration (MIC) against P.aeruginosa PAO1 , demonstrating a potent antibacterial activity. At MIC, the complexes were able to kill all the bacteria rather than simply inhibiting the growth (FIG. 2B), indicating the value of minimum bactericidal concentration (MBC) was equal to MIC. Thus, the complexes can be defined as bactericidal agents rather than bacteriostatic, as the MBC / MIC ratio is less than 4. Compared to the free Ag+and Fe2+combination, the selected Complex 3 exhibited a similar inhibitory effect as shown in FIG. 2C, showing that the complexation did not hamper the antibacterial activity of the ions at a 24-hr treatment duration. Moreover, single metal ion complexes, i.e., Fe complex and Ag complex, were also prepared for comparison (Table 3 and FIG. 8). The ratio was selected if the resulting solution is homogenous (Fe complex 3 and Ag complex 3). Fe complex hardly had antibacterial activity (MIC>256 pg / mL) and Ag complex (MIC= 16 pg / mL) demonstrated less efficacy compared to free Ag+and Fe@Ag complex. These results further supported the synergistic effect of these two ions. In addition to the potency of killing bacteria, the ability to avoid the emergence of resistance was also crucial for a bactericidal agent. To study the in vitro development of resistance, the bacteria was stimulated by sublethal concentrations of complex or the combination of free Ag+and Fe2+ions, and bacteria with visible growth at the highest concentration were sub-cultured daily. As shown FIG. 2D, no significant change in MIC was observed for both complex and free ions after 20 generations. This superiority might benefit from the dual mechanism of action of the combination of silver and iron.

[0124] Table 2. MICs of the complexes (uq / mL)

[0125] Complex Fe2+Cone. Ag+Cone. FIC

[0126] 1 2.2 8.4 1 .06

[0127] 2 2.9 5.6 0.71

[0128] 3 5.6 2.1 0.28

[0129] 4 8.3 1.6 0.23

[0130] 5 17.2 0.7 0.15

[0131] Fe2+alone 256 — —

[0132] Ag+alone — 8 —

[0133] Table 3. Preparation and characterization of Fe Complex and Ag Complex

[0134] Complex PA: ion Stability Size (nm) Zeta (mV)

[0135] Molar ratio Intensity mean

[0136] ' Fe-1 ' 1 :1 Turbid ' 4488 ' +2.95

[0137] Fe-2 2:1 Turbid 2562 +1.13

[0138] Fe-3 4:1 Homogenous 317 -27.5

[0139] Fe-4 8:1 Homogenous 170 -31.7

[0140] Ag-1 0.5:1 Colloidal 449 -49.8

[0141] Ag-2 1 :1 Colloidal 364 -52.4

[0142] Ag-3 2:1 Homogenous 344 -53.0

[0143] Ag-4 4:1 Homogenous 306 -53.3 Table 4. Antibacterial Activity of Fe Complex and Ag Complex

[0144] Complex Fe2+Cone Ag+Cone

[0145] Fe Complex >256 —

[0146] Ag Complex — 16

[0147] Fe2+alone 256 —

[0148] Ag+alone — 8

[0149] Example 3: In vitro release of ions

[0150] Overnight culture of P.aeruginosa and E.faecalis in LB broth was centrifuged (4000xg, 5 min), and the supernatant was collected and treated with equal volume of complex. For negative control, equal amount of complex was added with LB broth, with no bacteria. The mixture was incubated at 37°C 0.5, 1 , 3, 6, and 24 h. Then the released ions were collected by ultrafiltration using Amicon® Ultra-4 Centrifugal Filter Units (100k MWCO). Finally, the concentrations of ions were measured by ICP-OES.

[0151] Results and Discussion:

[0152] In the present design, the complex serves as a metal ion delivery platform that protects the ions from releasing until reaching the infection site, as the copolymer can be degraded by lipase, an extracellular enzyme secreted by many strains of bacteria, and among which Pseudomonas bacterium is regarded as a prolific producer. Therefore, the ability of the ions to release from the complex in the presence or absence of lipase-producing bacteria is vital to understanding the mechanism of the antibacterial activity. To study the release profile, the complex was incubated with P.aeruginosa supernatant or blank culture media as control. Another strain, E.faecalis, was also used as a lipase-negative control. The released amount was determined by ICP-OES after ultrafiltration. As shown in FIG. 3A, Fe2+underwent rapid release within 30 minutes of incubation with P. aeruginosa supernatant. In contrast, Ag release followed a more gradual kinetic profile, with over 75% of complexed silver being released within six hours of bacterial supernatant treatment, ensuring the antibacterial activity. This difference in release rates corresponded to previous findings that Ag / phosphate complexes (log Kc = 2.34) were more stable than Fe / phosphate complexes (log Kc = 2.23), and polymeric chelator systems could further magnify this affinity (K. Abu-Shandi, F. Al-Wedian, Estimation of composition, coordination model, and stability constant of some metal / phosphate complexes using spectral and potentiometric measurements, Chemical Papers 63(4) (2009) 420-425; J. Qian, C. Berkland, Conformational Stability Effect of Polymeric Iron Chelators, iScience 21 (2019) 124-134). By contrast, only a small amount of Fe and Ag releases were detected in blank medium and E.faecalis supernatant, indicating a good stability under a complex but lipase-free environment.

[0153] The bactericidal nature of the Ag-Fe complex was further verified and studied by time-killing experiments, where bacteria and different agents were co-incubated in growth media before viable cell numbers, represented by colony-forming units (CFUs), over time were determined. The complex achieved a 100% killing of an initial 107CFUs / mL bacteria at 3 hours and 1 hour under concentrations of 1 x and 2xMIC, respectively (FIG. 3C). At 0.5xMIC, although the number of viable bacteria decreased in the first hour, they recovered to grow afterward and reached the same amount as those treated by PBS (OxMlC, negative control), which was consistent with the incompetency at 0.5xMIC shown in FIG. 2B. The combination of free Ag+and Fe2+ions exhibited a similar trend in the time-killing curve but was faster, given the ion concentrations were the same (FIG. 3D). The difference in the killing rate between complex and free ions corresponded with the release profile, suggesting the killing effect was attributed to the metal ions release from the complex. In comparison, neither free Fe2+nor Ag+ion was able to eliminate the bacteria in the same range of concentrations (FIG. 3E and FIG. 3F).

[0154] Example 4: Mechanism Study

[0155] Log phase P.aeruginosa (1 x107CFU / mL) was incubated with an equal volume of complex (concentration equals to half of MBC) or free ions with the same ion concentrations for 30 min. The bacterial cells were collected by centrifugation at 4000xg for 5 min and washed with PBS. Then the cells were labelled with 5 pg / mL propidium iodide (PI) for 20 min, or 1 pM BODIPY- C11 for 30 min, or 10 pM DCFH-DA for 30 min to study the membrane permeability, lipid peroxidation, and ROS generation, respectively. Then the cells were washed with PBS twice and were detected by flow cytometry.

[0156] Results and Discussion:

[0157] It was confirmed the ions could be released from the complex, but how the liberated ions kill the bacteria remained unclear. Since it was reported that silver ions could increase bacterial membrane permeability (J.R. Morones-Ramirez, J. A. Winkler, C.S. Spina, J.J. Collins, Silver enhances antibiotic activity against gram-negative bacteria, Sci Transl Med 5(190) (2013) 190ra81 ), whether the complex could achieve it was verified by treating the bacteria with sublethal concentrations of different agents and then measuring the membrane integrity by propidium iodide (PI), a membrane-impermeable dye. As illustrated in FIG. 4A, all Ag+-, Ag++ Fe2+-, and complex-treated bacteria showed raised PI signals compared to PBS- and Fe2+- treated cells, indicating the increases in membrane permeability. Ferroptosis is an iron- dependent form of regulated cell death driven by lipid peroxidation, which occurs when lipid hydroperoxides accumulate in cellular membranes. While initially identified in mammalian cells, recent studies suggest that certain bacteria can also undergo ferroptosis-like death, primarily due to iron-mediated oxidative stress and membrane damage (X. Shen, R. Ma, Y. Huang, L. Chen, Z. Xu, D. Li, X. Meng, K. Fan, J. Xi, X. Yan, H. Koo, Y. Yang, J. Jiang, L. Gao, Nanodecocted ferrous polysulfide coordinates ferroptosis-like death in bacteria for anti-infection therapy, Nano Today 35 (2020) 100981 ; Z. Wang, H. Li, W. Zhou, J. Lee, Z. Liu, Z. An, D. Xu, H. Mo, L. Hu, X. Zhou, Ferrous sulfate-loaded hydrogel cures Staphylococcus aureus infection via facilitating a ferroptosis-like bacterial cell death in a mouse keratitis model, Biomaterials 290 (2022) 121842; W. Zhu, J. Mei, X. Zhang, J. Zhou, D. Xu, Z. Su, S. Fang, J. Wang, X. Zhang, C. Zhu, Photothermal Nanozyme-Based Microneedle Patch against Refractory Bacterial Biofilm Infection via Iron-Actuated Janus Ion Therapy, Adv Mater 34(51 ) (2022) e2207961 ; M.S. Kwun, D.G. Lee, Ferroptosis-Like Death in Microorganisms: A Novel Programmed Cell Death Following Lipid Peroxidation, J Microbiol Biotechnol 33(8) (2023) 992-997). To determine whether complex-induced bacterial killing exhibits characteristics of ferroptosis-like death including lipid peroxidation and ROS accumulation, BODIPY-C11 , a fluorescent probe that detects lipid peroxidation by shifting its emission from reduced form (581 / 591 nm) to oxidated form (488 / 510 nm) upon oxidation, was used. Following treatment with the complex or free Ag++ Fe2+, there was a notable decrease in reduced form (FIG. 4B) and a corresponding increase in oxidated form (FIG. 4C) compared to controls treated with PBS, Ag+alone, or Fe2+alone. These observations suggest an elevated lipid peroxidation and a potential induction of ferroptosis-like death pathways in bacteria by the complex. It is noteworthy that Fe2+treatment did not increase lipid peroxidation as much as the combination, indicated by a lower fluorescence intensity in the oxidized form (FIG. 4C). This observation might be attributed to the slower rate of iron ions alone to penetrate the intact bacterial cell to activate the lipid peroxidation process. But together with Ag+, which could increase the membrane permeability, the Fe ions could more efficiently enter the cells and enhance the antibacterial performance of Ag+by the activation of lipid peroxidation. These findings could explain the synergism of silver and iron. As a result, the intracellular level of ROS was increased in bacteria treated with all Fe2+, Ag+, free ions combination, and the complex, which was evidenced by the 2',7'-Dichlorofluorescein diacetate (DCFH-DA) staining shown in FIG. 4D, which finally induced cell death.

[0158] Example 5: Transcriptomic analysis

[0159] Log phase P.aeruginosa (1 x107CFU / mL) was incubated with an equal volume of complex (concentration equals to half of MBC) or free ions with the same ion concentrations for 30 min. The bacterial cells were collected by centrifugation at 4000xg for 5 min and washed with PBS. Then the total RNA was extracted by a FastPure Cell / Tissue Total RNA Isolation Kit V2-RC112

[0160] For RNA sequencing, 1 pg total RNA was used for following library preparation. The poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second-strand cDNA were synthesized. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor- ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Then libraries with different indexes were multiplexed and loaded on an Illumina HiSeq / Illumina Novaseq / MGI2000 instrument for sequencing using a 2x150 paired-end (PE) configuration according to manufacturer’s instructions.

[0161] Results and Discussion:

[0162] To thoroughly examine the impact on bacterial responses to the treatment, RNA sequencing (RNA-seq) was employed to evaluate the alterations in mRNA levels in P.aeruginosa subjected to Fe@Ag complex treatment. Compared to the PBS control, there were 1058 differentially expressed genes (DEGs) observed in the complex-treated group, where 417 genes were upregulated, and 641 genes were downregulated (FIG. 4E). Among these DEGs, genes with the highest fold changes and statistical significance were labelled in FIG. 4E and their functions were demonstrated in Table 5. Genes with functions related to cation transport (mexQ, copA1 , mex F), bacterial membrane (mexE, mexP) were significantly upregulated and genes with functions regarding iron transport (icmp, feoB, etc.) were significantly downregulated. In addition, gene ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) enrichments were performed to study the affected gene functions and signalling pathways. GO is an internationally recognized system to classify genes and their functions into molecular functions, cellular locations, and biological processes, and KEGG identifies enriched metabolic pathways. Top 20 GO enrichment revealed genes with functions of membrane function and ion transport were the most differentially expressed (FIG. 4F). And in KEGG enrichment (FIG. 4G), the pathway regarding the two-component system and APT-binding cassette (ABC) transporters were mostly affected. It has been reported that two-component system is used by P.aeruginosa io sense environmental Fe (II) (N.N. Kreamer, F. Costa, D.K. Newman, The Ferrous Iron-Responsive BqsRS Two-Component System Activates Genes That Promote Cationic Stress Tolerance, mBio 6(2) (2015) 10.1 128 / mbio.02549-14), and ABC transporter could actively transport metal ions (A.A. Akhtar, D.P. Turner, The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: Therapeutic and vaccine potential, Microb Pathog 171 (2022) 105734). The RNA-seq results indicated the complex treatment interfered with both membrane function and ion transport, which further validated the abovementioned antibacterial mechanism of the complex.

[0163] Table 5. Functions of significantly uprequlated and downrequlated qenes

[0164] Gene name -logic Log2(Fold Function

[0165] (Rvalue) Change) mexQ >300 9.26 Cation / multidrug efflux pump [Defense mechanisms] copA1 293 6.87 Cation transport ATPase [Inorganic ion transport and metabolism], mexE 238 5.56 Membrane-fusion protein [Cell envelope biogenesis, outer membrane] hypothetical 202 5.16 Copper chaperone [Inorganic ion protein-1 transport and metabolism]

[0166] Up- regulated opmE 172 8.25 Outer membrane protein [Cell envelope biogenesis, outer membrane / Intracellular trafficking and secretion] mexP 172 9.33 Membrane-fusion protein [Cell envelope biogenesis, outer membrane] hypothetical 167 6.20 CopZ, Copper chaperone [Inorganic ion protein-2 transport and metabolism] mexF 156 4.84 Cation / multidrug efflux pump [Defense mechanisms] icmp 108 -4.46 iron-regulated protein [Inorganic ion transport and metabolism] feoB 90 -4.67 Fe2+transport system protein B

[0167] [Inorganic ion transport and metabolism]

[0168] Down- regulated hypothetical 84 -5.43 ABC-type Fe3+transport system, protein-3 periplasmic component [Inorganic ion transport and metabolism] hypothetical 71 -5.08 Fe2+transport system protein A protein-4 [Inorganic ion transport and metabolism] Example 6: In vitro cytotoxicity assay

[0169] Mouse embryonic fibroblast 3T3 cell line was used as a mammalian cell model to test the cytotoxicity of the complex. And the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin / streptomycin), at 37 °C with 5% CO2. After the 80% confluence was reached, the cells were collected by trypsin seeded to 96-well plates with 1 x 104cells per well and cultured for 24 h before adding different concentrations of compounds. After another 24-h incubation, the cell viability was tested by Cell Counting Kit-8 (CCK-8).

[0170] Example 7: In vivo safety test

[0171] The animal studies were carried out in accordance to the protocols A21037 and A24047 approved by the NTU Institutional Animal Care and Use Committee (NTU-IACUC). BALB / c mice (8-9 weeks of age) were housed for 7 days in a 12h dark-light cycle at room temperature before any handling. The mice were anaesthetized with an intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg), then treated with PBS, free ions (Ag+1 mg / kg, Fe2+2.58 mg / kg), and the complex (same ion concentration as free ions) by intranasal (LN.) administration. Their body weights were recorded daily for 14 days. Mice with weight loss over 20% would be euthanized as humane endpoint.

[0172] To measure the ion concentrations in organs, the mice were euthanized after 3 h and 24 h post-administration of free ions and complex. The lungs and livers were collected, homogenized, and mixed with aqua regia for 24 h. Then the mixtures were filtered and the filtrates were collected to quantify the metals by ICP-OES.

[0173] For hematoxylin and eosin (H&E) staining, the mice were euthanized after 3 days postadministration of PBS, free ions and complex. The lungs and livers were collected and soaked in 4% paraformaldehyde for 24 h, 20% sucrose for 24 h and 30% for 24 h. Then the tissues were sectioned at 12 pm by freezing microtome, and stained with haematoxylin and eosin and mounted on glass slides.

[0174] Results and Discussion:

[0175] The safety profile of the metal ions could also be improved through the complexation strategy. FIG. 5A shows the viability of NIH / 3T3 cell line, a model mammalian cell line, after 24-hour incubation with the complex or the combination of free Ag+and Fe2+ions (free ions). The complex exhibited minimal toxicity to the cells compared to the free ions, as the ions could not be released from the complex in the absence of bacterial lipase, thereby enhancing the safety of such a system. The toxicity of single metal ions was also investigated as shown in FIG. 9A and FIG. 9B, and it was found that silver, not iron, was the source of toxicity. In contrast, neither single Ag complex nor Fe complex showed adverse effects to the cells (FIG. 9C and FIG. 9D), further confirming the enhanced in vitro safety of the complexation. To study the in vivo safety, healthy Balb / c mice were I.N. administered with PBS control, free ions, and the complex. Their bodyweight was recorded daily and the mice would be euthanized once the weight loss exceeded 20% as a human endpoint. As shown in FIG. 5B, all three groups experienced a drop in body weight on the 1 st day after administration. However, the free ion group continued to drop, and all the mice had over 20% weight loss by day 5. By contrast, mice received with complex gradually recovered during the experiment and achieved a comparable level as PBS control after 14 days. Furthermore, concentrations of silver and irons were measured at 3-h and 24-h post-administration to study the distribution and retention in lungs and livers. As shown in FIG. 5C-5E, both silver and iron concentrations in lungs decreased over time. However, free ions were cleared faster than the complex and the concentration was significantly lower compared to the complex group. It has been reported that particles with sizes less than 500 nm are more likely to be deposited in the alveoli by diffusion mechanism (I.M. El-Sherbiny, N.M. El-Baz, M.H. Yacoub, Inhaled nano- and microparticles for drug delivery, Glob Cardiol Sci Pract 2015 (2015) 2), and those larger than 500 nm could be cleared rapidly by mucociliary clearance (Z. Deng, G.T. Kalin, D. Shi, V.V. Kalinichenko, Nanoparticle Delivery Systems with Cell-Specific Targeting for Pulmonary Diseases, Am J Respir Cell Mol Biol 64(3) (2021 ) 292-307). In addition, particles with negative charges and PEG-modified surfaces could exhibit a higher likelihood of evading macrophage clearance. This explains the higher retention of our complex nanoparticles in the lungs, which is crucial for therapeutical efficacy. Furthermore, both silver and iron concentrations in livers increased (FIG. 5E and FIG. 5F), indicating a liver clearance mechanism. H&E staining was then conducted to study the histological changes after different treatments (FIG. 5G). Pathological changes were observed in lungs (lung edema, alveolar wall thickening, larger alveolar space) and livers treated by free ions. Whereas the complex group remains normal tissue morphology as PBS control, which further supported the enhanced safety profile of the complex.

[0176] Example 8: In vivo therapeutical efficacy study

[0177] For the antibacterial efficacy assay, each mouse was challenged with 20 pL of P.aeruginosa by I.N. administration (1 x10sCFU / mL) to establish acute lung infection. At 2- and 4-hours post-infection, the mice were treated with PBS, free ions (Ag+1 mg / kg, Fe2+2.58 mg / kg), and the complex (same ion concentration as free ions) by I.N. inoculation (n=5). At 24-h postinfection, the mice were euthanized by CO2 inhalation before the lungs and livers were collected and homogenized by a tissue homogenizer. The bacterial loads in the organs were measured by a traditional agar plate count method. To study the survival status, another group of mice were infected with 2x107CFU / mL P.aeruginosa. At 2- and 4-hours post-infection, the mice were treated with PBS, free ions (Ag+1 mg / kg, Fe2+2.58 mg / kg), and the complex (same ion concentration as free ions) by LN. inoculation (n=5). The body weight and survival status were recorded daily. Mice with weight loss over 20% would be euthanized as humane endpoint and would be recorded as death.

[0178] Results and Discussion:

[0179] The in vivo therapeutical efficacy of the complex was investigated through an acute bacterial pneumonia model, as P.aeruginosa is a common cause of hospital -acquired pneumonia. A single dose of PBS, free ions, and complex were given after I.N. administration of non-lethal dosage of P.aeruginosa. At 24h post-infection, the lungs and livers were harvested and the bacterial loads were counted. As illustrated in FIG. 6A and FIG. 6B, both the free ion and complex could significantly alleviate the bacterial burden in lungs and livers compared to PBS control. And there is no statistical difference in the antibacterial potency between free ions and the complex. Moreover, another set of mice were infected by lethal dosages of bacteria, and their body weight and survival status were recorded after different treatments. Although free ions exhibited potent in vitro and in vivo bactericidal activity, they could not save the mice from severe body loss (FIG. 6C), and all the mice experienced sharp weight loss in the first 3 days and were considered dead. Comparatively, one out of five mice treated with the complex could not be rescued from weight loss, while the other four mice survived throughout the 14- day observation period and their body weight gradually recovered (FIG. 6C and FIG. 6D). Taken together, these findings demonstrated the complex was safe for I.N. administration and was effective in treating in vivo lung infections.

[0180] In summary, the copolymer-based complex that addressed the limitations in metal ion delivery was successfully developed. This strategy leveraged the phosphonic acid-functionalized copolymer for robust coordination, ensuring the encapsulation of metal ions in physiological conditions. And the PCL component endowed the system with stimuli-responsive release of ions for selective antibacterial activity. These features were translated to effective in vitro and in vivo antimicrobial performance, particularly in the context of P. aeruginosa-induced pneumonia, a clinically relevant infectious disease. Importantly, the system maintained therapeutic efficacy without apparent toxicity, underscoring its potential for translational application in the field of metal ion drug delivery. Although this study focused specifically on Fe2+and Ag+, the copolymer provides a highly adaptable coordination environment that is not limited to these ions. As such, this delivery system is envisioned to evolve into a versatile platform technology for metal ion-based therapeutics.

Claims

CLAIMS1 . An antimicrobial composite material, comprising: a bacterial-enzyme-degradable anionic copolymer; and a plurality of metal ions, wherein the bacterial-enzyme-degradable anionic copolymer and the plurality of metal ions form a complex.

2. The antimicrobial composite material according to Claim 1 , wherein the plurality of metal ions comprise metal ions that can form two or more (e.g. 2, 3, 4, 5 or 6) ionic bonds with the bacterial-enzyme-degradable anionic copolymer.

3. The antimicrobial composite material according to Claim 1 or Claim 2, wherein the plurality of metal ions are selected from one or more of the group consisting of Ag+, Fe2+, Fe3+, Cu2+, Ni2+, Mn2+, Co2+, Zn2+, Ga3+, Ca2+, Mg2+, lr3+, Ce3+, and Ce4+.

4. The antimicrobial composite material according to any one of the preceding claims, wherein the plurality of metal ions comprise Ag+and Fe2+, optionally wherein the molar ratio of Ag+to Fe2+is from 0.01 :1 to 5:1 , such as from 0.02:1 to 2:1 , such as from 0.1 :1 to 1 :1 , such as about 0.2:1.

5. The antimicrobial composite material according to Claim 4, wherein the complex formed between the bacterial-enzyme-degradable anionic copolymer and the plurality of metal ions provides nanostructures.

6. The antimicrobial composite material according to any one of the preceding claims, wherein the antimicrobial composite material is in the form of particles, having a mean hydrodynamic diameter of from 100 to 500 nm, such as from 125 to 300 nm, such as from 155 to 291 nm, such as from 166 to 208 nm, such as about 175 nm.

7. The antimicrobial composite material according to any one of the preceding claims, wherein the molar ratio of the bacterial-enzyme-degradable anionic copolymer to the collective total of metal ions is from 1 :1 to 5:1 , such as from 1 :0.67 to 4:1 , such as about 3.33:1 .

8. The antimicrobial composite material according to any one of the preceding claims, wherein the bacterial-enzyme-degradable anionic copolymer has the formula (I):where:R is a Ci to C linear or branched alkylene chain;A represents C=O or P(ORa)=O;Rarepresents a linear or branched Ci to Cg alkyl group; a represents a block polyethylene glycol unit having a number average molecular weight of from 500 to 10,000 Daltons; b and c together represent a random copolymer block, where b and c each independently have a value of from 8 to 100.

9. The antimicrobial composite material according to Claim 8, wherein one or more of the following apply:(a) a represents a block polyethylene glycol unit having a number average molecular weight of about 2,000 Daltons;(b) b and c each independently have a value of from 10 to 50, such as 15 to 20, such as 18;(c) A represents C=O; and(d) R represents -(CH2)5-.

10. The antimicrobial composite material according to Claim 8 or Claim 9, wherein the bacterial-enzyme-degradable anionic copolymer has a number average molecular weight of from 6,000 to 10,000 Daltons, such as from 6,900 to 8,000 Daltons.11 . The antimicrobial composite material according to any one of the preceding claims, wherein the bacterial-enzyme-degradable anionic copolymer is degradable by a bacterial- secreted lipase or a bacterial-secreted phosphoesterase, such as a bacterial-secreted lipase.

12. The antimicrobial composite material according to any one of the preceding claims, wherein the antimicrobial composite material has a zeta potential of from -10 to -60 mV, such as from -34 to -52.1 mV, such as from -36.4 to -49.6 mV, such as about -39.8 mV.

13. A pharmaceutical composition comprising an antimicrobial composite material according to any one of Claims 1 to 12 and one or more of a pharmaceutically acceptable excipient and carrier.

14. An antimicrobial composite material according to any one of Claims 1 to 12, or a pharmaceutical composition as described in Claim 13, for use as a medicament.

15. A method of treating a subject suffering from a microbial and / or fungal infection comprising the steps of administering to the subject a therapeutically effective amount of an antimicrobial composite material according to any one of Claims 1 to 12, or a pharmaceutical composition as described in Claim 13, such that the infection is treated.

16. Use of an antimicrobial composite material according to any one of Claims 1 to 12, or a pharmaceutical composition as described in Claim 13, in the manufacture of a medicament to treat a microbial and / or fungal infection in a subject in need thereof.

17. An antimicrobial composite material according to any one of Claims 1 to 12, or a pharmaceutical composition as described in Claim 13, for use in the treatment of a microbial and / or fungal infection.

18. A composition comprising an antimicrobial composite material according to any one of Claims 1 to 12, optionally wherein the composition is a cosmetic composition or a detergent composition.

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