A method for preparation of silver-gallium composition and its uses

Abstract

A composition of matter comprising silver-gallium (Ag-Ga) amalgamated particles is disclosed. A coating comprising silver-gallium (Ag-Ga) amalgamated particles is also disclosed. The composition or coating may be antibacterial.

Description

A METHOD FOR PREPARATION OF SILVER-GALLIUM COMPOSITION AND ITS USESPRIORITY DOCUMENT

[0001] The present application claims priority from Australian Provisional Patent Application No. 2023903102 titled “A METHOD FOR PREPARATION OF SILVER-GALLIUM COMPOSITION AND ITS USES” and filed on 26 September 2023, the content of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to antimicrobial materials and coatings.BACKGROUND

[0003] Biomedical device-associated infections (BDAIs) pose a significant global challenge for both patients and healthcare systems.[1]Despite the implementation of stringent disinfection management and aseptic surgical environments, BDAI infection rates remain between 2-10% in developed countries.[2]Due to inequalities in healthcare resources and practices, infection rates in developing nations can escalate up to 15%.[3]BDAIs can lead to severe complications, including disability, mortality, and greatly increased healthcare expenditure. There is an urgent need for enhanced infection prevention measures, improved resource accessibility, and innovative interventions to alleviate the burden of these infections on a global scale.

[0004] Traditional antibiotic therapies have limitations in effectively resolving BDAIs, as relapse after treatment remains a substantial concern. The high risk-infections such as these in the case of bone implants are closely linked to patients with compromised immune systems and the formation of biofilms on the implant surfaced6, 8]The latter require significantly higher doses of antibiotics, up to 1000-fold, for effective treatment.[9]Hence, there is an urgent need for innovative solutions that can help reduce device colonization by bacteria and establishing a long-term protection against infections.

[0005] The antibacterial properties of silver (Ag) have been well-established and utilized in various medical devices, including wound dressings such as Acticoat (Smith&Nephew),

[0010] Tegaderm,

[0011] GranuFoam Silver,

[0012] and catheters like Bardex Silver,

[0013] Palindrome™,

[0014] and ARROW® CVCs.

[0015] The growing challenge of antibiotic -resistant bacteria, coupled with the positive outcomes observed with Ag- coated dressings and catheters, has stimulated further research and development efforts to incorporate silver into orthopedic implants. Recent advancements have led to the market introduction of Ag-coated megaprostheses, including METS Silver Agluna® System (Stanmore Implants), MUTARS® (Implantcast GmbH), and PorAg® (Waldemar Link), demonstrating promising results in reducing infection rates.

[0016] However, there are persistent challenges in addressing early inflammatory responses and enhancing the integration of these implants with bone tissue (osteointegration).

[0017] Furthermore, emerging reports of bacterial resistance to silver raise concerns about the long-term effectiveness of Ag-based antimicrobial treatments.

[0018]

[0006] In particular, it has been observed that repeated exposure to silver nanoparticles (AgNPs) can induce resistance in Gram-negative bacteria, such as various strains of Escherichia coli, without involving genetic alterations.118 191This resistance is attributed to phenotypic changes that result in the suppression of flagellum protein synthesis. Investigations have shown that strains resistant to AgNPs did not simultaneously develop resistance to Agions, suggesting that bacterial cells may respond differently to distinct forms of silver.

[0020] It is noteworthy that certain bacteria possess specific genes, e.g. the sil genes (e.g., sil , silP, and silS), which contribute to resistance against Ag ions by facilitating efflux, sequestration, or reduction of silver.

[0021] Further research is required to unravel the underlying mechanisms of Ag resistance and provide alternative or complementary strategies to combat this threat.

[0007] There is thus a need to provide antibiotic materials or coatings that ameliorate of overcome one or more of the problems associated with known antibiotic materials or coatings. Alternatively, or in addition, there is a need to provide antibiotic materials or coatings that provide a useful alternative to known antibiotic materials or coatingsSUMMARY

[0008] According to a first aspect, there is provided a composition of matter comprising silver-gallium (Ag-Ga) amalgamated particles.

[0009] In certain embodiments, the silver-gallium (Ag-Ga) amalgamated particles comprise from about 0.01 at. % to about 70 at. % silver in the Ag-Ga amalgamed particles, such as about 1.5, about 3, about 4 or about 6 at. % silver in Ag-Ga amalgamed particles. In certain specific embodiments, the silver-gallium (Ag-Ga) amalgamated particles comprise about 6 at. % silver in Ag-Ga amalgamed particles.

[0010] In certain embodiments, the composition of matter further comprises a surfactant. The surfactant may be a poloxamer such as a Pluronic™ surfactant. In certain specific embodiments, the Pluronic™ surfactant is Pluronic™ F-127.

[0011] In certain embodiments, the Ag-Ga amalgamed particles are nanoparticles. The nanoparticles may have an average diameter of from about 1 nm to about 350 nm.

[0012] The silver-gallium (Ag-Ga) amalgamated particles are formed by galvanic deposition of silver nanocrystals on an oxide layer of gallium liquid metal (GaLM).

[0013] According to a second aspect, there is provided a solution comprising the silver-gallium (Ag-Ga) amalgamated particles of the first aspect. The solution may be an aqueous solution, such as a solution in water.

[0014] According to a third aspect, there is provided a coating comprising the silver-gallium (Ag-Ga) amalgamated particles of the first aspect.

[0015] According to a fourth aspect, there is provided a device comprising the coating of the third aspect. In certain embodiments, the device is a medical device.

[0016] According to a fifth aspect, there is provided an antimicrobial composition comprising the silver-gallium (Ag-Ga) amalgamated particles of the first aspect.

[0017] According to a sixth aspect, there is provided an antimicrobial coating comprising the silver- gallium (Ag-Ga) amalgamated particles of the first aspect. In certain embodiments, the coating comprises from about 0.05 pg Ag / cm2to about 300 pg Ag / cm2. In certain embodiments, the coating comprises from about 0.1 pg Ga / cm2to about 300 mg Ga / cm2.

[0018] Advantageously, the antimicrobial is antibacterial in certain embodiments. In certain specific embodiments, the coating achieves 99% elimination rates for S. aureus and P. aeruginosa at concentrations of 16 pg Ag / cm2and 176 pg Ga / cm2.

[0019] According to a seventh aspect, there is provided a device comprising the antimicrobial coating of the sixth aspect.

[0020] According to an eighth aspect, there is provided a process for producing silver-gallium (Ag-Ga) amalgamated particles, the process comprising mixing a solution comprising Ag+with a suspension comprising Ga particles under conditions to form silver-gallium (Ag-Ga) amalgamated particles.

[0021] In certain embodiments, the process comprises forming the suspension comprising Ga particles by subjecting a suspension of gallium in water to sonication. In certain embodiments, the sonication is for a period of from about 15 seconds to about 60 minutes, such as from about 15 minutes to about 35 minutes. In certain embodiments, the sonication is for a period of about 25 minutes.

[0022] In certain embodiments, the suspension of gallium in water is cooled during the sonication.

[0023] In certain embodiments, the conditions to form silver-gallium (Ag-Ga) amalgamated particles comprise galvanic deposition of silver nanocrystals in the oxide layer on gallium liquid metal (GaLM).

[0024] In certain embodiments, the process further comprises treating the silver-gallium (Ag-Ga) amalgamated particles with a surfactant.

[0025] In certain embodiments, the surfactant is a poloxamer. In certain specific embodiments, thepoloxamer is a Pluronic™ surfactant. In certain of these specific embodiments, the Pluronic™ surfactant is Pluronic™ F-127.

[0026] According to a ninth aspect, there is provided silver-gallium (Ag-Ga) amalgamated particles produced by the process of the eighth aspect.BRIEF DESCRIPTION OF THE FIGURES

[0027] Embodiments of the present disclosure will be discussed with reference to the accompanying figures wherein:

[0028] Figure 1 is a schematic illustration of the formulation of an Ag-Ga nano-amalgamated particles suspension according to embodiments of the present disclosure, and application on medical devices;

[0029] Figure 2 shows (a) A schematic depiction of the preparation of an Ag-Ga nanoparticle suspension according to embodiments of the present disclosure, (b) The morphology of gallium particles including gallium oxide coating (outside) and gallium liquid metal (inside) in solution, (c) Schematics of galvanic replacement GaLM particles with Ag ion. (d) The Ag-Ga formulation coated by F-127. SEM micrograph of Ga particles (e) and Ag-Ga suspension with 1.5% Ag (f) and 6% Ag (g), scale bar is 1 pm. (h) Energy dispersive X-ray spectroscopy (EDX) cross-section mapping of 6% Ag in Ag-Ga suspension synthesized from direct probe sonication, scale bar is 2 pm. TEM image of Ga particles (i) and Ag-Ga suspension with 6% Ag (j), showing the presence of Ag and the smooth spherical shape of the Ga particles; (k) high-resolution TEM (HRTEM) image and (1) its fast Fourier transform (FTT) image, revealing the detection of silver structure in the suspension, scale bars are 200 nm and 2.5 nm. (m) the contribution sizes of Ga particles and Ag-Ga (6 at. % Ag). (i) The XRD findings indicate the presence of Ag nanoparticles, as evidenced by the diffraction peaks associated with the (111), (200), (220), and (311) planes;

[0030] Figure 3 shows SEM images of unwashed (a) and washed (b) Ag-Ga nano-amalgam particles with various concentrations of AgNCL;

[0031] Figure 4 shows XPS spectra of the chemical composition of Ag-Ga suspension showing the presence of chemical elements in the Ag-Ga synthesis with different silver concentrations;

[0032] Figure 5 shows zones of inhibition were generated by the diffusion of different concentrations of Ga, Ag+, Ag-Ga and AgNPs on .S'. aureus (a) and P. aeruginosa (b). Data illustrates mean ± sd, n = 3;

[0033] Figure 6 shows antibacterial properties of Ag-Ga particles. Log reduction of Ag ion, Ag-Ga and Ga on S. aureus (a) and P. aeruginosa (b) after 24 hours incubation. Error bar represents mean ± s.d, n = 3, ***p < 0.001, **p < 0.01, *p < 0.05. (c) CLSM and SEM images of S. aureus following 24 h incubation on the surfaces coated Ga (176 pg / cm2) and Ag-Ga (16 pg Ag + 176 pg Ga / cm2). (d) CLSMand SEM images of P. aeruginosa following 24 hours incubation on the surfaces coated Ga (176 pg / cm2) and Ag-Ga (16 pg Ag + 176 pg Ga / cm2). Scale bars are 10 pm (CLSM) and 1 pm (SEM). (e) The concentrations of silver ion and gallium ion release from Ag+, Ag-Ga and Ga in TSB media after 24 hours incubation at 37 °C. Values represents mean ± s.d, n=3. To explore the practical application, the Ag-Ga suspension was sprayed onto surfaces inoculated with S. aureus (f) and P. aeruginosa (g), which revealed no growth of either bacterium in the sprayed areas;

[0034] Figure 7 shows ROS staining images of P. aeruginosa and S. aureus (a) after incubating with surfaced coated Ag-Ga, Ag+and Ga for 24 hours. The H2DCFDA (green, kex= 480 nm, kem= 525 nm) fluorescence dyes were used for ROS detection. The membrane potential of P. aeruginosa and S. aureus were taken after a 24-hour incubation with surface-treated Ag-Ga, Ag+, and Ga. The fluorescence from the DOI dye (kex= 484 nm, kem= 501 nm) and the fluorescence from the DPA dye (kem= 406 nm) were employed to detect membrane potential;

[0035] Figure 8 shows bacterial growth inhibition by spraying method. Ag+, AgNPs and Ga solution were sprayed and incubated at 37 °C for 24 hours on agar plates cultured with P. aeruginosa (a) and S. aureus (b);

[0036] Figure 9 shows principal component analysis (PCA) of untreated and treated S. aureus cells. Comparative PCA score plots (left side) and PCA loading spectra correlations PC-1 (right side) for S. aureus treated by Ag-Ga (a) and Ga (b) with S. aureus. The left side displays the comparative PCA score plots, whereas the right features the PCA loading spectra correlations for PC-1, which illustrates comparison of P. aeruginosa when treated with Ag-Ga (c) and Ga (d) to untreated P. aeruginosa',

[0037] Figure 10 shows evaluating the cytotoxicity and wound healing ability of the coatings containing Ga, Ag+, Ag-Ga. CCK8 assay assessing the viability of HaCaT cells (a) incubated in DMEM with Ag / Ag-Ga for 24 hours, (b) CLSM images of HaCaT cells following 24 hours incubation with silicon surfaces coated Ag-Ga, Ag+and Ga (Green: Phalloidin; Blue: DAPI). (c) Cell migration assay of Ag and Ag-Ga conditioned medium and control medium for 0 hours, 24 hours and 48 hours, demonstrating increased cell motility and proliferation in the case of cells incubated in Ag-Ga conditioned medium and control medium. Mean ± s.d, n = 3, ***p < 0.001, **p < 0.01, *p < 0.05;

[0038] Figure 11 shows in vivo antibacterial activity of Ag-Ga surface-coating implants, (a) Basic workflow of the animal experiments, (b) Intraoperative process of model building, (c) Representative images of external and internal surface of skin where the implant was located, (d) The changes in the ordinary blood test, (e) Representative images of H&E staining, Masson staining, and Giemsa staining of skin surrounded the implants. Scale bars, 200pm. (f) The Immunohistochemical results of inflammatory markers COX-2, INOS, and macrophage marker CD68. Scale bars, 200pm (low field), 50 pm (high field), (g) Statistical analysis for IOD of COX-2, INOS, and CD68 based on immunostaining result. Errorbar represents mean ± s.d.; n = 3, ***p < 0.001, **p < 0.01, *p < 0.05;

[0039] Figure 12 shows representative images of H&E staining of the major organs;

[0040] Figure 13 shows in vivo anti-infective assessment in an MRSA-induced rat femoral osteomyelitis model, (a) Schematic illustration of the MRSA-injected femoral osteomyelitis model, (b) Gross investigations of rat femurs at specified times, (c) Representative X-ray images, (d) Representative images of Micro-CT images, (e) Quantitative results of bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N) in the ROI. Error bar represents mean ± s.d.; n = 3, ***p < 0.001, **p < 0.01, *p < 0.05;

[0041] Figure 14 shows statistical analysis for trabecular separation (Tb.Sp) (a), and trabecular thickness (Tb.Th) (b) of Micro-CT results;

[0042] Figure 15 shows histological assay of MRSA-induced rat femoral osteomyelitis model, (a-c) Representative photos of H&E staining, Giemsa staining, and Masson staining to evaluate the inflammatory infiltration. Scale bars, 200pm. (d) Representative images of Immunohistochemical staining of iNOS in different indicated groups. Scale bars, 200pm (low field), 50 pm (high field), (e) Statistical analysis for IOD of INOS based on immunostaining result, (f-g) Immunohistochemical staining of COX-2. Scale bars, 200pm (low field), 50 pm (high field), (h) COL-1 immunohistochemical staining of the representative implant was performed from different groups. Scale bars, 200pm (low field), 50 pm (high field), (i) Statistical analysis for IOD of COL-1 based on immunostaining result, ( j-k) Immunohistochemical staining of TGF-[3L Scale bars, 200pm (low field), 50 pm (high field). Scale bars, 200pm (low field), 50 pm (high field). Error bar represents mean ± s.d.; n = 3, ***p < 0.001, **p < 0.01, *p < 0.05; and

[0043] Figure 16 shows in vivo promoting osteogenic ability in rat critical-size calvarial defect model, (a) Schematic illustration of calvarial defect establishment treatment modalities, (b) Intraoperative process of model building, (c) Representative 3D reconstruction of Micro-CT images, (d) Representative images of H&E staining, and (e) Masson staining Scale bars, 500pm (low field), 100 pm (high field), (f) Immunohistochemical staining of BMP-2, BMPR, and Runx-2 positive cells in different groups, (g) Statistical analysis for IOD of BMP-2, BMPR, and Runx-2 based on immunostaining result. Error bar represents mean ± s.d.; n = 3, ***p < 0.001, **p < 0.01, *p < 0.05.DESCRIPTION OF EMBODIMENTS

[0044] The following detailed description describes certain embodiments and is not to be taken in a limiting sense.

[0045] All weights, amounts and ratios herein are by weight, unless otherwise specifically noted. Theterms shown below have the following meanings:

[0046] As used herein unless otherwise specified, the term “antimicrobial” describes a biocidal effect that may be, for example, an antibacterial, antifungal, antiviral, bacteriostatic, disinfecting, or sanitising effect.

[0047] As used herein unless otherwise specified, the term “antibacterial” means having the ability to cause greater than a 90% numeric reduction in a population of one or more of Staphylococcus aureus, Pseudomonas aeruginosa or any other infective bacteria.

[0048] The present disclosure arises from the inventors’ research using gallium liquid metal (GaLM) particles as carriers for silver to achieve in a synergistic silver-gallium (Ag-Ga) nano-amalgamated particles through the galvanic replacement process. Surprisingly, the so-formed silver-gallium (Ag-Ga) nano-amalgamated particles show enhanced antimicrobial properties and low toxicity.

[0049] Thus, described herein is a composition of matter comprising silver-gallium (Ag-Ga) amalgamated particles.

[0050] Also disclosed herein is a process for producing silver-gallium (Ag-Ga) amalgamated particles, the process comprising mixing a solution comprising Ag+with a suspension comprising Ga particles under conditions to form silver-gallium (Ag-Ga) amalgamated particles.

[0051] The synthesis process transforms Ag+into Ag nanocrystals through galvanic replacement, with silver nanocrystals robustly adhering to the GaLM particle surfaces. Thus, the conditions to form silver- gallium (Ag-Ga) amalgamated particles comprise galvanic deposition of silver nanocrystals in the oxide layer on gallium liquid metal (GaLM).

[0052] The gallium (Ga) component of the silver-gallium amalgamated particles is formed from any suitable form or source of gallium. For example, gallium liquid metal is readily available commercially. Gallium liquid metal listed here includes gallium alloys, including gallium-indium alloys, galliumindium- tin alloys. The suspension comprising Ga particles may be formed in water or other suitable aqueous solution. The amount of gallium added to water to make a suspension may be from about 0.1 mg / mL to about 10 mg / mL. In some exemplary embodiments, the amount of gallium added to water is from about 5 mg / mL to about 6 mg / mL such as 5.7 mg / mL.

[0053] Gallium has emerged as a promising alternative for various medical applications due to its unique properties and associated advantages. Previous studies have demonstrated that Ga exhibits moderate antimicrobial activity against a wide range of pathogens, making it a versatile solution for addressing bacterial infections.

[0022] Importantly, Ga ions possess a distinct mechanism of action that disrupts bacterial iron metabolism, thereby reducing the potential for the development of bacterialresistance. Clinical trials involving gallium nitrate have shown efficacy in treating bacterial infections in human subjects.

[0023] Notably, Ga nanoparticles can induce anti-inflammatory responses without interfering with the iron homeostasis pathway in immune cells. Also, Ga nanoparticles were demonstrated to possess low cytotoxicity,

[0024] while gallium nitrate and citrate were found to promote bone growth.

[0025]

[0054] The silver (Ag) component of the silver-gallium amalgamated particles can be formed from any suitable silver precursor. For the galvanic deposition of silver nanocrystals in the oxide layer on gallium liquid metal, a source of silver ions (Ag+) can be used. Suitable sources of silver ions include silver salts such as, but not limited to, silver nitrate (AgNOs), silver sulfate (Ag2SO4), silver sulfide (AgzS), silver tetrafluoroborate (AgBF4), silver perchlorate (AgC104), silver cyanate (AgOCN), silver iodide (Agl), silver bromide (AgBr), silver chloride (AgCl), silver hexafluorophosphate (AgPFe), silver nitrite (AgNOz), as well as organic silver salts, such as silver acetate and silver citrate.

[0055] The solution comprising Ag+can be any aqueous solution such as water. For example, the solution comprising Ag+can be any of the aforementioned silver salts. Solutions of silver nitrate in water at a concentration of from about 0.1 mg / mL to about 20 mg / mL can be used. An aqueous silver nitrate solution comprising 10 mg / mL silver nitrate can be used.

[0056] The silver-gallium amalgamated particles are formed from a suspension comprising Ga particles produced by subjecting a suspension of gallium in water to sonication. The sonication can be for any suitable period from about 15 seconds to about 60 minutes, such as from about 15 minutes to about 35 minutes, such as 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes or 35 minutes. In certain embodiments, the sonication is for a period of about 25 minutes.

[0057] The suspension of gallium in water may be cooled during the sonication. Cooling can be by any suitable means, such as, for example, cooling the suspension with ice, running water, or cooled solvent. Cooling during sonication may minimise the formation of crystalline gallium oxide monohydroxide (GaOOH) in the sonication process.

[0058] The silver-gallium (Ag-Ga) amalgamated particles may be stabilised by treating them with a surfactant. It is contemplated that any suitable surfactant could be used. For example, the surfactant may be a polyoxyethylene sorbitan monooleate or a poloxamer. Poloxamer surfactants are particularly suitable. Poloxamers are non-ionic triblock copolymers composed of a central hydrophobic chain of poly oxypropylene flanked by two hydrophilic chains of polyoxyethylene. Suitable poloxamer surfactants are available under the trade name Pluronic™ and include, but are not limited to, F127, F38, F 108, F68, F77, F87, F88, F98, L101, L121, L61, L62, L63, L81, L92, P103, P104, P15, P123, P65, P84, and P85. For example, Pluronics™ which may be suitable for use include, but are not limited to, F127 and F87.For example, Pluronic™ F-127 may be used. One of these surfactants may be used alone, or two or more thereof may be used in combination.

[0059] The Ag-Ga amalgamed particles may comprise from about 0.01 at. % to about 70 at. % silver or from about 1 at. % to about 10 at. % silver, such as about 1 at. %, about 1.5 at. %, about 2 at. %, about 2.5 at. %, about 3 at. %, about 3.5 at. %, about 4 at. %, about 4.5 at. %, about 5 at. %, about 5.5 at. %, or about 6 at. % silver. For example, the Ag-Ga amalgamed particles may comprise about 1.5 at. %, about 3 at. %, about 4 at. % or about 6 at. % silver. Ag-Ga amalgamed particles comprising about 6 at. % silver may be particularly suitable.

[0060] The Ag-Ga amalgamed particles are nanoparticles. The average diameter of the Ag-Ga amalgamed particles may be from about 1 nm to about 350 nm, such as from about 100 nm to about 500 nm.

[0061] The silver-gallium (Ag-Ga) amalgamated particles may be produced in an aqueous solution, such as a water solution.

[0062] Advantageously, GaLM demonstrates an ability to markedly mitigate silver toxicity through forming silver nanocrystals on the GaLM oxide surface, while amplifying antimicrobial potency, attenuating inflammation, and bolstering osseointegration. In vivo evaluations disclosed herein validated the robust antibacterial properties, including eliminating pathogens such as methicillin-resistant S. aureus and E. coli. Furthermore, Ag-Ga particle coatings support bone-mimetic matrix.

[0063] As demonstrated herein, the silver-gallium (Ag-Ga) amalgamated particles have antimicrobial properties. In particular, the silver-gallium (Ag-Ga) amalgamated particles have antibacterial properties. For example, as disclosed herein, a coating comprising the silver-gallium (Ag-Ga) amalgamated particles achieves 99% elimination rates for S. aureus and P. aeruginosa at concentrations of 16 pg Ag / cm2and 176 pg Ga / cm2.

[0064] Also as demonstrated herein, the silver-gallium (Ag-Ga) amalgamated particles have antiinflammatory properties.

[0065] Also as demonstrated herein, the silver-gallium (Ag-Ga) amalgamated particles have osseointegration properties by promoting collagen development.

[0066] The silver-gallium (Ag-Ga) amalgamated particles may be part of a coating. The coating may be applied to a device. For example, a coating comprising the silver-gallium (Ag-Ga) amalgamated particles may be provided on a non-medical device such as a building material, a construction material, a sealant, etc. In these applications, the coating may provide antimicrobial properties to the device, such as antibacterial properties.

[0067] Alternatively, a coating comprising the silver-gallium (Ag-Ga) amalgamated particles may be provided on a medical device such as an implant, a wound dressing, etc. The medical device can be formed from a material such as ceramic, plastic, magnesium alloy or titanium. In these applications, the coating may provide antimicrobial properties, anti-inflammatory properties and / or osseointegration properties to the medical device.

[0068] The Ag-Ga particle suspension can be applied to a device using any suitable coating method such as spray coating, dip coating or other coating technology. For example, the Ag-Ga particle suspension can be applied to a device by spray coating. Following spray coating, the coating may be dried to form a stable surface coating comprising silver-gallium (Ag-Ga) amalgamated particles.

[0069] The amount of silver and gallium in the coating can be formulated using suitable relative amounts to gallium and Ag+when producing the particles. For example, the coating may comprise from about 0.05 pg Ag / cm2to about 300 pg Ag / cm2, such as from about 5 pg Ag / cm2to about 30 pg Ag / cm2and / or from about 0.10 pg Ga / cm2to about 300 mg Ga / cm2, such as from about 100 pg Ga / cm2to about 300 pg Ga / cm2.

[0070] Optionally, a composition or a coating comprising the silver-gallium (Ag-Ga) amalgamated particles may further comprise an additional antimicrobial agent. The additional antimicrobial agent may be sodium pyrithion, zinc pyrithion, an additional silver salt such as silver nitrate, silver sulfadiazine, povidone iodine, active oxygen compounds such as hydrogen peroxide, isolated or equilibrium derived or isolated peracids such as chloroperbenzoic acids, peracetic acid, perheptanoic acid, peroctanoic acid, perdecanoic acid, performic acid, percitric acid, perglycolic acid, perlactic acid, perbenzoic acid, and monoester peracids derived from diacids or diesters such as adipic, succinic, glutaric, or malonic acid; amphenicols; ampicillins; ansamycins; beta-lactams such as carbacephems, carbapenems, cephalosporins, cephamycins, monobactams, oxacephems, penicillins and any of their derivatives; carboxylic esters such as p-hydroxy alkyl benzoates and alkyl cinnamates; chitosan salts; cubic-phase lipids; additional gallium- containing antimicrobial agents such as gallium acetylacetonate, gallium bromide, gallium chloride, gallium fluoride, gallium iodide, gallium maltolate, gallium nitrate, gallium nitride, gallium percolate, gallium phosphide and gallium sulfate; iodo-compounds and other active halogen compounds such as iodine, interhalides, polyhalides, metal hypochlorites, hypochlorous acid, metal hypobromites, hypobromous acid, chloro- and bromo-hydantoins, chlorine dioxide and sodium chlorite; lincosamides; macrolides; nitrofurans; organic peroxides including benzoyl peroxide and alkyl benzoyl peroxides; ozone; phenolic derivatives including o-phenyl phenol, o-benzyl-p-chlorophenol, tert-amyl phenol and Ci-Ce alkyl hydroxy benzoates; quaternary ammonium compounds such as alkyldimethylbenzyl ammonium chloride and dialkyldimethyl ammonium chloride; quinolines; singlet oxygen generators; sulfonamides; sulfones; sulfonic acids such as dodecylbenzene sulfonic acid; tetracyclines; vancomycin; derivatives thereof and mixtures thereof. Many of these recited agents represent classes containing useful specific materials whose individual utility will be recognized by persons having ordinary skill in the art.For example, exemplary penicillins include, but are not limited to, amdinocillin, amdinocillin pivoxil, amoxicillin ampicillin, apalcillin, aspoxicillin, axidocillin, azlocillin, acampicillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, carbenicillin, carindacillin, clometocillin, cloxacillin, cyclacillin, dicloxacillin, epicillin, fenbenicillin, floxacillin, hetacillin, lenampicillin, metampicillin, methicillin sodium, mezlocillin, nafcillin sodium, oxacillin, penamecillin, penethamate hydriodide, penicillin G benethamine, penicillin G benzathine, penicillin G benzhydrylamine, penicillin G calcium, penicillin G hydrabamine, penicillin G potassium, penicillin G procaine, penicillin N, penicillin O, penicillin V, penicillin V banzathine, penicillin V hydrabamine, penimepicycline, phenethicillin potassium, piperacillin, pivampicillin propicillin, quinacillin, sulbenicillin, sultamicillin, talampicillin, temocillin, ticarcillin and mixtures thereof or with other materials.EXAMPLES

[0071] Preparation of Ag-Ga suspension: A solid gallium piece (~57 mg) was added to 10 mL of water and sonicated for different times from 15 to 35 minutes in a standard probe sonication (Vibra-Cell, Sonics) with an ice bath to form a Ga particles suspension. Pluronic F-127 (Sigma-Aldrich) was dissolved in water to make 10% solution and silver nitrate (Sigma- Aldrich) was dissolved in water to make a solution at concentration of 10 mg / ml. Different concentrations of Ag-Ga nano-amalgamated particle suspension were prepared by adding silver nitrate solution into the Ga particles suspension, and then mixing using a vortex mixer before the addition of F-127 (Figure 1 and Figure 2a) in the ratio silver atomic percentage of 0%, 1.5%, 3.0%, 4.0%, 6.0% respectively.

[0072] To prepare thin films for testing, aliquots of 20 pL, 40 pL, 80 pL and 160 pL of each sample were pipetted into wells of 96 well-plates and dried at room temperature. The thin films thus formed were used for antimicrobial and viability testing.

[0073] Scanning Electron Microscopy (SEM) Characterization: The surface morphology and chemical composition of Ag-Ga synthesis were examined using SEM (FEI Inspect F50). The synthesized suspensions were imaged at 3 kV and the images were analyzed using ImageJ software. The chemical composition of Ag-Ga representing in scanning region and underlying of the suspension was examined by EDS spectra. For bacteria cellular imaging, Ag-Ga and Ga suspensions were deposited on silicon wafers. P. aeruginosa and S. aureus cultures (OD~0. 1) were then placed on silicon wafer coated with Ga / Ag-Ga and incubated at 37 °C for 18 hours. Bacterial cells were also incubated on the silicon wafer to serve as control. After the incubation period, the bacterial cells were fixed with glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4, 4% (ProSciTech, Australia) for 45 minutes. The cells were then dehydrated with a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 10 minutes for each concentration. The samples were air-dried and coated with a thin platinum film using an Ion Sputter Coater (TB-SPUTTER, Quorum Technologies, UK) before imaging.

[0074] X-ray photoelectron spectroscopy characterization: Surface chemistry was assessed via X-ray photoelectron spectroscopy (XPS), obtaining spectra for Ag 3d, Ga 2d, C Is, and O Is. A Thermo Scientific K-alpha XPS spectrometer with an Al Ka X-ray source (1486.7 eV) and a surrounding hemispherical electron analyzer were utilized. Samples of Ga, Ag-Ga, and Ag were set on silicon bases. The acquired XPS data were processed using the CasaXPS software, referencing the C Is peak at 285 eV for charge.

[0057]

[0075] Transmission Electron Microscopy (TEM) Characterization: A JEOL JEM-2100F transmission electron microscope (TEM) was operated at an acceleration voltage of 200 kV to capture low- and high- resolution TEM images of the samples. The TEM samples were prepared by depositing dilute solutions of Ga and Ag-Ga suspensions onto TEM grids. The TEM images were obtained and analyzed using the Gatan Digital Micrograph software package.

[0076] X-ray crystallography (XRD): After producing Ga and Ag-Ga droplets, samples were washed with water and drop-cast onto glass slides and allowed to dry. XRD patterns were taken with a Bruker D4 diffractometer utilizing Cu Ka radiation (1.5418 A). The collected data was then processed to create line charts using the Origin 2023 software.

[0077] Inductively coupled plasma mass spectrometry (ICP-MS): To evaluate the release of Ga and Ag ions from the Ga-F127, Ag-Ga-F127, and Ag-F127 samples, these were submerged in 0.5 mL of TSB media maintained at 37 °C for 24 hours. Once the incubation period finished, the supernatants were collected and filtered to ensure any impurities or granules were removed. This liquid underwent treatment with HNOs to break down any residual solids and was subsequently diluted to keep the concentration under 5%, this is to minimize complications during ICP-MS evaluations. The Varian 720-ES system, America, was employed to measure Ga and Ag ion content in the liquid using ICP-MS. Calibration of this device was done using reference solutions of specified concentrations. The ion content in the original samples was ascertained using a comparison curve derived from these reference solutions.

[0078] Bacterial strains and culture: The bacterial strains, specifically Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 15692, were maintained at -80 °C. To initiate the experiments, the acquired bacterial strains were streaked on tryptic soy agar (TSA, Oxoid) plates. A single colony was selected from each plate, suspended in 10 mL of sterilized Tryptic Soy Broth (TSB, Oxoid), and incubated at 37 °C with shaking for 18 hours before being used for the experiments.

[0079] Antibacterial Studies: Antibacterial activity of Ag-Ga suspension was assessed against S. aureus and P. aeruginosa using the CFU counting method. Briefly, 100 pL of bacteria cultures (106CFU / mL), S. aureus and P. aeruginosa, were added to 96-well plate containing samples as prepared herein and incubated for 24 hours at 37 °C. After incubation period, serial dilution of 1:10 (v / v), 1:100 (v / v) and 1:1000 (v / v) were prepared from the overnight cultures and 5 pL of each dilution was spreadonto tryptic soy agar (TSA) plates. The plates were incubated for 18 hours at 37 °C and the number of colonies was counted. Log reduction was calculated and compared to control samples (untreated bacteria). The experiment was conducted in triplicate.

[0080] Well diffusion assay: Bacteria cultures (106CFU / mL) were spread on TSA plates. Wells (5 mm) were cut into the agar plates with a sterilized cork borer and then 50 pL of Ag-Ga suspension was pipetted into the wells. AgNCL. Ga and AgNPs solutions (50 pL) were also poured into each well for antibacterial property comparison. Inoculated plates were then incubated at 37 °C for 24 hours for zone inhibition.

[0081] Live and dead staining: After incubation bacterial on the silicon wafers coated Ag, Ag-Ga and Ga, samples were rinsed twice with (PBS) and then stained with the LIVE / DEAD® BacLightTM Viability Kit (containing SYTO®9 and propidium iodide, PI) at room temperature for 10 minutes in the dark. The samples were observed using a ZEISS LSM 880 microscope (Zeiss, Germany). All experiments were performed in triplicate.

[0082] Membrane potential staining: Following the incubation of bacteria on silicon wafers coated with Ag, Ag-Ga, and Ga, each sample was subjected to two washes with Phosphate-Buffered Saline (PBS). The samples were then stained with 1 pM of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), supplied by Invitrogen, Australia, and left at room temperature for 10 minutes in a dark setting. Following another wash with PBS, these samples were analyzed using a ZEISS LSM 880 microscope, manufactured by Zeiss, Germany.

[0083] Reactive Oxygen Species staining: Post-incubation of bacteria on silicon wafers that had been coated, the samples underwent two washes with Phosphate-Buffered Saline (PBS). Following this, samples were treated with 1 pM of DiO stain from Biotium Corp., Hayward, CA, USA, and left to sit in darkness for 5 minutes. Subsequently, 2 pM of DPA was introduced to the bacterial mixture and allowed to incubate for another 45 minutes in a dark environment. Finally, fluorescence imagery was acquired using Confocal Laser Scanning Microscopy (CLSM).

[0084] Synchrotron ATR-FTIR Operation and Data Analysis: The spatial distribution of chemical functional groups in both untreated and treated bacteria with Ag-Ga, Ga, and Ag was studied using ATR- FTIR mapping. This research took place at the Infrared Micro-spectroscopy beamline of the Australian Synchrotron, utilizing a Bruker Hyperion 2000 FTIR microscope linked to a VERTEX V80v FTIR spectrometer from Bruker Optik GmbH in Ettlingen, Germany. We used a distinct macro ATR-FTIR equipment which included a 100 mm diameter germanium (Ge) ATR crystal (with a refractive index of nGe= 4.0) and a 20x IR objective (with a numerical aperture of NA = 0.60). Pathogen bacteria were exposed to Ag-Ga, Ga, and Ag for a duration of 3 hours at a temperature of 37 °C. After air-drying, both the untreated and treated samples were positioned on aluminum discs, which were then placed on thesample stage of the macro ATR-FTIR instrument. Using the OPUS 8.0 software from Bruker, spectral parameters such as a range between 3900-950 cm1and a resolution of 4 cm1was set. This software also allowed to create chemical maps by calculating the area under specific peaks in the spectral data. For in- depth multivariate data analysis, we utilized CytoSpec v. 1.4.02 from Cytospec Inc. in Boston, MA, USA, and the UnscramblerX 11.1 software by CAMO Software AS, located in Oslo, Norway.

[0085] To clarify the biochemical modifications in the bacteria strain affected by Ga and Ag-Ga nanoamalgam, we used ATR-FTIR micro-spectroscopy. This approach enabled us to detect variations in the bacteria's structural molecules, which were evident in the spectral data gathered from the Synchrotron macro ATR-FTIR micro-spectroscopy system.

[0086] To examine the biochemical interactions between Ga and Ag-Ga nano-amalgam and both Gram-negative and Gram-positive bacterial cells, we gathered detailed maps using ATR-FTIR microspectroscopy synchrotron technology. Our focus was specifically on understanding molecular alterations within the bacterial cell membrane and nucleus. For this, we targeted crucial spectral zones, such as the methylene region (3000-2800 cm1) for lipid changes, the amide region (1705-1600 cm ') for protein alterations, and a specific range (1200-950 cm ' ) for modifications in polysaccharides and nucleic acids.

[0087] Using the collected spectral information, we generated tables that depict these important spectral regions. These tables can be interpreted in terms of the FTIR absorption intensity, which was calculated by integrating the data over the specified spectral ranges

[0059] .

[0088] The visual representations of the spectral maps offer insights into the distribution of functional groups within the targeted bacteria. Recognizing the variations in the spectra corresponding to these groups aids in assessing the structural alterations in the bacteria post-exposure to Ga and Ag-Ga nanoamalgam.

[0089] To statistically analyse the variations in the spectral maps, a hierarchical cluster analysis (HCA) was carried out to categorize regions with analogous spectral features. We focused our analysis on specific spectral ranges that included protein, polysaccharide, and nucleic acid (1810-1000 cm1), as well as lipid (3004-2810 cm1) spectra. To emphasize the key spectral areas, the second derivative, which serves as an indicator for the overall rate of spectral change, was applied as noted in previous studies.

[0060] Then, five spectral plots rooted in the average spectral values from clusters were created that displayed similar spectral characteristics. To identify the predominant average spectra for use in principal component analysis (PC A), the cluster(s) were evaluated and selected, that covered the largest area within the spectral map. Additionally, the chosen average cluster spectrum was required to have both the highest peak intensities and the lowest noise levels among functional groups. This included groups like amide I and -CH stretching, which are known for having notably distinct peaks useful for cell identification. These criteria were applied to pick a cluster likely containing cells, thereby facilitating comparisonsbetween samples before and after treatment.[59b> 61]

[0090] To examine and highlight significant variations in the spectral readings taken by the synchrotron macro ATR-FTIR system across untreated and treated samples, HCA was utilized, as previously mentioned. Before subjecting the spectra to extended multiplicative signal correction (EMSC), every spectrum within the selected cluster was first processed through a second-derivative transformation. Following this preliminary step, the compiled set of spectra was subjected to Principal Component Analysis (PC A) to probe similarities and differences among the cellular populations, as cited in prior research.

[0061] The PCA values corresponding to the primary components, namely PC-1 and PC-2, were computed and illustrated in Figure 5 and Figure 9. These calculated values were used to shed light on the differences between cell samples before and after being treated.

[0091] Mammalian cell culture: Human epidermal keratinocyte cells (HaCaT, 300493, cell line services, Eppelheim, Germany) and human fibroblast cells (HFF-1 SCRC-1041, USA) were used in this study. The cells were stored in liquid nitrogen until reviving using the ATCC standard protocol. Briefly, 1 mL of frozen cells were thawed in a 37 °C water bath and then diluted with 5 mL of fresh Dulbecco’ s modified Eagle’s medium (DMEM) (for adherent cell) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Gibco, Thermo Fisher Scientific). The mixture was centrifuged at 200 ref for 5 minutes and the pellet was resuspended in fresh media and incubated in humidified air atmosphere containing 5% CO2 at 37 °C in either T25 or T75 flasks (Corning, NY). The media was changed every 2- 3 days, and the cells were sub-cultured when they reached the confluency approximately 80%. The HaCaT cells were detached from the flask, using 0.25% Trypsin-EDTA (Gibco, Thermo Fisher Scientific) before conducting any experiments. Cell number and viability were determined using a trypan blue exclusion test with an automated Countess III Cell Counter (Thermo Fisher Scientific). Before conducting any experiments, the cells were grown for at least 1 week after thawing to ensure stability and viability.

[0092] Cell viability assay: Cell viability was assessed via a cell counting kit-8 (CCK8) assay (Ab228554, Abeam, Australia). Briefly, 100 pL of cells in media were added to 96-well plate containing samples as prepared in section of preparation of Ag-Ga suspension and incubated at 37 °C with 5% (v / v) CO2 atmosphere. The untreated wells with cells seeding were used as positive control for viability. Cells were seeded into the plates with density of IxlO5cells / well. After 24 hours of incubation, 10% of CCK8 solution was added to each well and the cells were incubated for 4 hours. The absorbances were measured at 460 nm using a BioTek Synergy HTX Multimode microplate reader. All assays were studied in triplicate.

[0093] Immunofluorescence staining: cells were fixed with 4% para-formaldehyde in phosphate buffered saline (PBS) (Sigma-Aldrich) for 20 minutes. Following fixation, cells were rinsed using PBS and then treated with a 0.1% Triton-X solution in PBS for 3 to 5 minutes, after which they were againwashed with PBS. Subsequently, the cells were exposed to a mixture of DAPI and phalloidin (both from Invitrogen, Life Technologies) in PBS and allowed to sit for 30 minutes at room temperature. Excess dye was then removed by another PBS wash. Finally, cell imaging was carried out using an Olympus 1X83 fluorescence microscope, with excitation wavelengths of 365 nm and 489 nm, respectively.

[0094] Cell Migration Assay: The IncuCyte SX5 live-cell analysis system (Sartorius) was utilized for the real-time scratch closure experiment. The human fibroblast (HFF) cells were seeded on 96-well plates at a density of IxlO5cells per well and incubated at 37 °C with 5% CO2 for 24 hours to allow cell attachment and growth. Scratch was created by consistently scraping the cells in each well using a wound maker. The detached cells were then removed by washing with PBS solution, and fresh growth medium (DMEM / F12 + containing 10% FBS, 100 units / mL penicillin and 100 pg / mL streptomycin) was added to the control wells. In the test wells, 100 pL of growth medium containing 10% of Ag-Ga or Ag suspension were added. The plates were incubated in the IncuCyte SX5 system for 48 hours at 37 °C with 5% CO2 and 95% humidity. Images were captured after 24 hours and 48 hours (period until the scratch wound was completely closed).

[0058] The width of the scratch was subsequently measured after 0 hours, 24 hours, and 48 hours using ImageJ software.

[0095] Animal studies: 10-Week-old male Sprague Dawley rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were housed under controlled identical specific pathogen free (SPF) standard environmental conditions (23 ± 2 °C, 12 hours light / dark cycle) with free access to food and allowed to move freely. All animal treatments and surgical procedures were conducted in accordance with the guidelines of the Ethics Committee on Animal Experiments of Shandong University in China (Approval No. 23027).

[0096] Subcutaneous bacterial infection model

[0097] The rats were anaesthetised by an intraperitoneal injection of 1% sodium pentobarbital at a dose of 40 mg / kg. Before implantation, the right hindlimb of the rat was treated with hair removal cream and disinfected with iodophor. Next, an incision was made in the right hindlimb of approximately 10 millimeters to form a subcutaneous pocket, and the NP-coated disc (diameter 8 millimeters) was inserted. Immediately after implantation, 1 x 106cfu of S. aureus or E. coli in 50 pl PBS were injected directly onto the disc surface to establish a subcutaneous bacterial infection model in rats. The incision was then closed with 3-0 silk thread sutures and disinfected.

[0098] Rat critical-size calvarial defect model

[0099] To prepare for bone transplantation implantation, a group of 20 animals received intraperitoneal injections of 1% sodium pentobarbital at a dosage of 40 mg / kg for anaesthesia. A stereotactic framework was used to stabilize the head, and a linear incision was made along the midline of the scalp to expose the skull. A skull defect with a diameter of 8 mm was created in the parietal bone using a micro drill,specifically centered around the sagittal suture line between lambda and the anterior chamber. The surgical site was continuously rinsed with physiological saline solution to reduce the risk of infection, and the discs were carefully placed into the bone defect (n=4). Finally, the wound was meticulously sutured and disinfected.

[0100] MRSA-induced rat femoral osteomyelitis model

[0101] The rats were anaesthetised by intraperitoneally injecting 1% sodium pentobarbital at a dose of 40 mg / kg, and the skins of their right hindlimbs were prepared for surgery. The knee joint of the rat was fixed in the maximum flexion position, and a 15-mm longitudinal incision was made along the lateral patella under sterile conditions. The dislocated knee joint was separated layer by layer to expose the femoral condyle. A 1-mm drill was then used to drill the center of the femoral condyle in the direction of the medullary cavity. Then, the bone debris was washed with saline, and 100 pl of 1 x 107CFU / mL bacterial suspension was injected into the femoral medullary cavity with a 1-ml syringe. Finally, the drill hole was sealed with bone wax, and the incision was closed layer by layer.

[0102] Micro-CT

[0103] Before histological processing, samples after paraformaldehyde fixing without decalcification were used for micro-CT. Micro-CT scanning was set at an isometric resolution of 36 pm, with X-ray energy settings of 70 kV and 200 pA. The microstructure shape of vertebrae was measured using a Quantum GX2 Micro-CT scanner (PerkinElmer, USA). The scanned images from each group were evaluated at the same thresholds to allow 3-dimensional (3D) structural reconstruction of each sample.

[0104] Histological analysis

[0105] Microscopic evaluation of the internal structure of the skin, skull, and the indicated femur was analyzed by histological analysis. Rat bones in each experimental group were separated and processed with 4% PFA for 72 hours, then decalcified in 10% w / v EDTA for 30 days, followed by dehydration and embedding in paraffin; subsequently, 5 pm thick sections were cut. Hematoxylin and eosin (H&E) staining Masson Trichrome staining as well as Giemsa staining was performed and imaged using a light microscope.

[0106] Immunohistochemistry

[0107] For immunohistochemical evaluation, the sections were performed with an Immunohistochemistry Kit (SP-9000, ZSGB-BIO, China) according to the instructions. After dewaxing and hydration, the sections were deparaffinized and incubated in 3% H2O2, then the sections were treated with Tris-ethylenediaminetetraacetic acid antigen retrieval buffer (C1038, Solarbio) for 10 minutes at 95 °C and endogenous peroxidase blocker for 10 minutes. After blocking in goat serum for 30 minutes atroom temperature, the cells were incubated with primary antibodies against COX-2 (1:200, Proteintech, China), INOS (1:200, Proteintech, China), CD68 (1:300, Proteintech, China), Col-1 (1:200, Proteintech, China), TGF-[3 (1:200, Abeam, USA), BMP-2 (1:200, Proteintech, China), BMPR (1:200, Proteintech, China), Runx-2 (1:100, CST, USA) at 4 °C overnight. Then, the sections were incubated with goat antirabbit immunoglobulin (IgG) secondary antibodies for 1 hour at room temperature and horseradish peroxidase (HRP)-labeled Streptomyces ovalbumin for 15 minutes. After rinsing with PBST, the slices were incubated with a DAB kit (ZSGB-BIO, China) for visualization. Then the slices were washed in tap water, counterstained with hematoxylin, dehydrated, and cover slipped with neutral resin. Images were taken with a panoramic digital slice scanning microscope (Olympus, Tokyo, Japan). Immunopositivity within the fields was calculated using Image J software (NIH, MA, USA).

[0108] Results and Discussion

[0109] Synthesis and characterization of Ag-Ga suspension

[0110] Silver-gallium (Ag-Ga) nano-amalgamated particles were synthesized by mixing Ga droplets suspension with AgNO? and F-127 solutions. Firstly, the Ga particle suspension was obtained using probe ultrasonication methods as previously described.

[0026] A suspension of gallium (Ga) was created by introducing around 57 mg of liquid gallium into 10 mL of Milli-Q (MQ) water. The mixture was then subjected to probe sonication for varying durations ranging from 15 to 25 minutes. Due to the effects of heat on the formation of crystalline gallium oxide monohydroxide (GaOOH) (rod-shape),[26a]probe sonication was performed in an ice bath, and then mixing with AgNO? using a vortex mixer before the addition of F-127 (Figure 2a). The structure of the gallium (Ga) particles, enveloped by an oxide layer, is schematically depicted in Figure 2b. Each Ga particle consists of a liquid Ga core surrounded by native surface oxides.

[0027] This unique composition gives the Ga particles an adhesive capacity, which can be attributed to the combined effects of the enveloping surface oxides and the malleability of the liquid core.

[0028] The size of the Ga particles (Table 1) reveal that as the ultrasonication period extends from 15 to 35 minutes, the Ga particles tend to be of reduced average sizes and exhibit a more uniform size spread. However, no notable change in size distribution was observed between the 25 to 35-minute mark.Therefore, the 25-minutue time point was selected for synthesizing GaLM for the experiments discussed later.

[0111] Table 1 - Dimensions ofGa and Ag-Ga particles for different sonication time points

[0112] The diagram shown in Figure 2c outlines the strategy for producing Ag-Ga through galvanic replacement. When silver nitrate solution is introduced to a Ga suspension, the Ag-Ga particles form spontaneously through the galvanic replacement process, represented by Equation (1).Ga(l) + 3Ag+(aq) 3Ag(s) + Ga3+(aq), E° = 1.328 V (1)

[0113] We propose, as depicted in Figure 2c, that when Ga particles interact with an AgNO? solution, silver nanocrystals emerge and become affixed onto the oxide layer of the GaLM droplets. This event can be better understood by referencing the theory of Mott-Cabrera on standard reduction potentials, detailing the chemical reaction typically present on metal oxides.

[0029] This concept suggests that metals inherently develop an approximately 3nm thick oxide layer on their exteriors, which forms spontaneously because of electronic transitions.

[0030] In our case, the reaction progression is likely due to the presence of thin metal oxide layer and the pronounced difference in standard reduction potentials between the electron donor (GaLM) and the electron acceptor (silver ions).

[0031] Hence, a reaction that is thermodynamically spontaneous transpires on the GaLM oxides surface, influenced by the standard reduction potential (with Ga3+ / Ga and Ag+ / Ag values being -0.529 and 0.799 V against the standard hydrogen electrode (SHE), respectively).

[0032] The outcome is the formation and attachment of silver crystal particles to the GaLM oxide surface.

[0114] Once the Ag-Ga particles have been generated, a modest quantity of F-127 is introduced to the solution to establish a stabilizing coating around the Ag-Ga particles. F-127 is a non-toxic polymer that has been approved by FDA for biomedical applications.

[0033] Extensively researched for its applications incellular and drug delivery, F-127 possesses advanced characteristics like minimal toxicity, temperature- triggered gelation in reverse, the ability to encapsulate drugs, and the capacity to form a gel even at low concentrations.

[0034] Therefore, we selected F-127 as a safe surfactant to maintain the stability of the Ag-Ga suspension as without it the Ga suspension would rapidly precipitate within 1 hour (Figure 2d).[26a]

[0115] To better visualise the Ag nanocrystals attached to the oxide layer of the Ga particles, the Ag- Ga particles were washed with water to remove excess F-127. Both, the Ga and Ag-Ga particles, were subsequently inspected using a Scanning Electron Microscope (SEM). The SEM images showed that the Ga only suspension produced mostly smooth and spherical particles (Figure 2e) while Ag-Ga suspension contained rough spherical particles with bound crystal structure (determined to be solid silver) on the surface of the Ga particles (Figure 2f). Interestingly, the interaction between the Ga particles and Ag ions appears to become more pronounced as the concentration of AgNO? increases. As evidenced in Figure 2g, the Ga particles have undergone substantial depletion, leading to the emergence of a pronounced surface indentation, and coupled with an increase in the crystalline particle count. Therefore, the structure of the Ga particles undergoes notable changes with increasing the concentration of silver ions (Figure 3).

[0116] The presence of Ag nanocrystals binding to Ga particles was further investigated using other techniques. Scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS) was utilized to determine the present of silver on the surface of the GaLM particles in the Ag-Ga nanoamalgam, showing the presence of Ag and Ga in the sample analysis (Figure 2h). Transmission electron microscopy (TEM) images of Ag-Ga revealed alterations in the Ga particles. In contrast to the uniform circular mass observed in Ga particles alone (Figure 2i), the configuration of Ga particles in Ag-Ga was no longer uniform or circular (Figure 2j). Furthermore, the substance within the gallium turns porous and exhibits reduced density, highlighting the changes in atomic concentration within the Ga particles after interaction with silver nitrate. Also, the distinction between the Ga droplets (larger particles) and the tiny nanoparticles adhering to the Ga particles surface is evident due to the variations in their atomic structures. Moreover, a high-resolution TEM (HRTEM) image and its relevant Fast Fourier-transform (FTT) images also revealed a d-spacing of 0.24 nm (Figure 2k), matching well with the (111) plane of face-centered cubic structure of silver (Figure 21).

[0035] The presence of solid silver was consistently verified using X-ray diffraction (XRD) techniques. In Figure 2m, the XRD patterns of Ag-Ga are presented, which were produced from the interaction of Ga droplets with Ag ions. Peaks identified at 10 values of 38.45°, 44.76°, 64.79°, and 77.72° align with the (111), (200), (220), and (311) planes of Ag crystals.

[0036] These peaks are indicative of a face-centered cubic (FCC) lattice formation and its inherent crystalline structure, as indexed in JCPDS-04-0783.

[0037] These observations align with the TEM findings, where Ag nanocrystals were observed to form on the surface of Ga particles. This provides compelling evidence that the nanoparticles found around Ga particles are silver nanocrystals, formed from the reaction between Ga (in liquid form) and AgNO? at 37 °C. We would use 6% Ag in Ag-Ga nano- amalgamed particles for our antimicrobial, biocompatibility, and in vivo evaluation.

[0117] X-ray photoelectron spectroscopy (XPS) was utilized to examine the elemental compositions and chemical states on the synthesized particle surfaces. Figure 4 displays the XPS results for Ga droplets with varying Ag concentrations (0 at. %, 1.5 at. %, 3 at. %, 4 at. % and 6 at. %). In all samples, except for pure Ga droplets, silver was discernible through doublet peaks, specifically Ag ds / 2 and Agat binding energies of 367.5 eV and 373.4 eV, respectively.

[0038] Gallium presence was marked by peaks at binding energies of 1118.5 eV and 1145.2 eV, corresponding to Ga lpj / 2 and Ga 2 pi / 2, respectively.

[0039] Importantly, the distinct signals from both silver and gallium elements were minimally affected by the Pluronic surfactant (F-127). This indicates that the polymer layer encasing the Ag-Ga might be thinner than 10 nm, the detection threshold of XPS. Furthermore, the C Is peak was deconvoluted into three distinct peaks. The dominant binding energy peaks at 286.5 eV and 285 eV correspond to the C-0 and C- C bonds, respectively, and were detected across all samples. These peaks confirm the incorporation of F- 127 on the particle surface. The O-C bond peak, found at 533 eV, had the highest intensity under the O Is peaks. Signal for the O-Ga bond was seen at binding energy of 531 eV. Collectively, the XPS data suggest that both Ga and Ag-Ga droplets are enveloped by a thin surfactant layer (F-127), which could potentially inhibit further gallium oxidation. The XPS results showed that the Ga and Ag-Ga droplets could be covered by a thin layer of F-127 which might have prevented the further oxidation of gallium.

[0118] Antimicrobial properties of Ag-Ga

[0119] The antibacterial activity of the Ag-Ga nano-amalgamated particles was assessed against Staphylococcus aureus and Pseudomonas aeruginosa. These represent two frequently encountered bacteria in BDAIs.

[0040] These bacteria also serve as representatives for Gram-positive and Gram-negative types and their distinct cellular structures. When examined via the disc diffusion method, the Ga particle suspensions exhibited a relatively weak antibacterial zone with diameters of 8.56 mm ± 0.40 and 7.23 mm ± 0.25 for P. aeruginosa and S. aureus, respectively, shown in Figure 5. The antibacterial potential was significantly amplified when Ga particles were reacted with AgNOs to form Ag-Ga nano-amalgamated particles. The inhibition zones formed by the Ag-Ga particles were 11.2 mm ± 0.25 for P. aeruginosa and 10.2 mm ± 0.26 for S. aureus. Additionally, as indicated in Figure 6a, b, Ag-Ga particles displayed an impressive efficiency in eradicating bacteria upon contact, achieving 99% elimination rates for both S. aureus and P. aeruginosa at concentrations of 16 pg Ag and 176 pg Ga / cm2. Notably, the antibacterial ability of Ga particles was substantially augmented, especially towards S. aureus when integrated with silver nanocrystals on the metal oxide exterior.

[0120] The viability of the pathogen cells on silicon wafers coated with Ag-Ga and Ga was accessed using confocal laser scanning microscopy (CLSM). The results are shown in Figure 6c. For this study, we employed Live / Dead fluorescent staining (with green signifying live cells and red for dead cells) following a 24-hour period of interaction with either the control or the nanoparticle coatings. In the case of S. aureus, only a slight reduction in the intensity of the green fluorescence (corresponding to live bacteria) was observed on the Ga particles coated surface. In comparison, near complete death of S.aureus cells was observed on the Ag-Ga coated surface, accompanied by intense red fluorescence denoting dead bacteria. On the other hand, the efficiency in eradicating P. aeruginosa by both the Ag-Ga and Ga coatings was nearly comparable (as depicted in Figure 6d). This can be attributed to the potent bactericidal effect of Ga against P. aeruginosa documented in the scientific literature.[22g, 41]

[0121] The antimicrobial action of Ag-Ga composite nanoparticles was investigated through membrane potential and Reactive Oxygen Species (ROS) assays. Figure 7a demonstrates that exposure to Ag-Ga coated surfaces led to membrane potential shifts in both tested pathogenic bacteria, evidenced by green fluorescence in their cells. These alterations can be attributed to the Ag-Ga nanoparticles adhering to the bacterial cell membrane, as depicted in SEM images of Figures 6c, d. Such adherence can disrupt the integrity of membrane, leading to wrinkling, tearing, and the formation of holes. This causes significant changes in bacterial cell morphology and membrane functions, ultimately leading to cell inactivation. Additionally, the antimicrobial properties of Ag-Ga are also due to its ability to generate ROS. These are highly reactive molecules and free radicals formed from molecular oxygen. As shown in Figure 7b, intracellular ROS were evident in both bacterial types when exposed to Ag-Ga coated surfaces. Notably, the fluorescent intensity of ROS in S. aureus was higher compared to that in P. aeruginosa. ROS can cause cellular damage by oxidizing DNA, RNA, carbohydrates, proteins, and lipids, which can lead to cell death.

[0042]

[0122] SEM imaging provided further support by demonstrating a reduced number of bacteria on both Ag-Ga and Ga coated surfaces. Notably, the Ag-Ga coating surface revealed pronounced damage to the cell membranes, visibly disrupting the original rod-like morphology of P. aeruginosa and the spherical morphology of S. aureus (Figure 6c, d). The nanoparticles significantly change the bacterial cell structure, resulting in cell deactivation. This finding aligns well with published studies, which show that bacterial deformation arose due to the adherence between the cell membrane and Ga particles.

[0043] Additionally, the disruption of bacterial morphology is notably enhanced when silver nanoparticles are present on the surface of the Ga particles. Collectively, the Ag-Ga combination demonstrates a heightened efficacy against S. aureus compared to Ga particles alone.

[0123] In the Ag-Ga system, Ga particles function as a reservoir for silver formed through a reaction between the Ga droplets and silver nitrate. This suggests a potential for a moderated release of silver ions into the surrounding environment. Indeed, based on the inductively coupled plasma MS (ICP-MS) analysis presented in Figure 6e, the release of silver ions from surfaces coated with Ag-Ga in a tryptic soy broth (TSB) environment over 24 hours at 37 °C was approximately 14 times lower than that from surfaces coated with Ag ion. Simultaneously, the release of gallium ions from both the Ga and Ag-Ga coated samples was similar, with concentrations above 3000 pm. As a result, the Ag-Ga system, given its inherent ability to encapsulate and release silver ions at a controlled rate, is not only proficient in eradicating bacterial cells but also can sustain this advantage for extended durations. Furthermore, theprolonged and calibrated release of silver ions could potentially mitigate the cytotoxic effect associated with high concentration of silver ions.

[0124] To evaluate the surface antibacterial efficacy of the Ag-Ga solution, an area of TSA (tryptic soy agar), pre-coated with the two types of pathogenic bacteria used in this work, was spray-coated with Ag- Ga particles and then incubated for 24 hours. It is worth highlighting that the regions treated with the Ag- Ga solution developed prominent antibacterial zones (Figure 6f, g). Conversely, those areas treated with Ga or AgNPs only do not possess clear antibacterial zones (Figure 8).

[0125] The intricate biochemical alterations instigated induced by Ag-Ga and Ga on bacteria.

[0126] We conducted synchrotron macro-attenuated total reflection (ATR)-FTIR micro-spectroscopy to investigate the intricate biochemical shifts triggered by Ag-Ga and Ga particles in two pathogenic bacteria, i.e. the Gram-positive Staphylococcus aureus and the Gram-negative Pseudomonas aeruginosa. This technique enabled us to identify shifts in the structural molecules of the bacteria. We conducted principal component analysis (PCA) to identify distinct changes in the ATR-FTIR spectra between bacteria treated with Ag-Ga and Ga suspensions, and untreated bacteria, as shown in Figure 9.

[0127] For S. aureus, biochemical modifications following interactions with Ga and Ag-Ga particles were evident through PC-1 loadings of 89% and 92% of the spectral variation, respectively. These data suggest that there were substantial biochemical shifts. Peaks associated with PC-1, utilized to determine the most distinct wavenumbers and their chemical components between treated and untreated bacteria, emerged from the divergence in PCA loading. The intensity of these loading peaks provides insight into the degree of difference, with heightened intensities reflecting greater variation. Central peaks are illustrated in Figures 9a&b. In Figure 9a, the recorded peak intensities span bands indicative of the lipid zone, amide I, and polysaccharides. This points to changes in chemical structures tied to bacterial membranes and proteins.

[0044] Changes of particular interest occur in the polysaccharide structures of bacteria, evidenced by the elevated intensity of peaks within this zone. Concurrently, the engagement of Ga particles with S. aureus resulted in notable shifts within the lipid and polysaccharide domains, as highlighted by the peak intensities in these sectors (Figure 9b). The potential of Ag-Ga and Ga particles to bring about biochemical structural changes was similarly seen in P. aeruginosa, with PC-1 loading values of 98% and 99%, respectively (Figures 9c, d). These shifts are discernible by the occurrence of peaks in regions associated with lipids, proteins, and polysaccharides. Notably, in the lipid area of both samples, two prominent groups of peaks corresponding to modifications in the methyl / methylene groups (2922 cm1and 2858 cm ' l of the phospholipids of cell membrane were detected.

[0045] These modifications are likely due to the cell membrane damage caused by the impact of Ga-Ag and Ga particles (SEM images in Figure 9c, d).

[0128] Biocompatibility and cell migration assays

[0129] To investigate the effects of Ag, Ga, and Ag-Ga suspensions on cell viability, we carried out the cell count kit -8 (CCK8) assay with different density of a suspensions (8 pg / cm2, 16 pg / cm2, 24 pg / cm2, 32 pg / cm2of Ag; 88 pg / cm2, 176 pg / cm2, 264 pg / cm2, 353 pg / cm2of Ga; Ag-Ga respectively) in HaCaT cells after 24 hours of incubation. As shown in Figure 10a, Ag significantly reduced cell growth at all densities of the suspension with a the rate of 11%; conversely, Ga slightly reduced cell growth with an acceptable rate (under 30%), according to ISO 109993-5 standards.

[0046] Surprisingly, Ag-Ga successfully reduced cell cytotoxicity of Ag from 11% in Ag group to 84% in Ag-Ga group.

[0130] Furthermore, confocal laser scanning microscopy (CLSM) was utilized to visualize cells incubated on Ag-Ga, Ag and Ga, and compared to controls (silicon wafers). Cells exposed to Ga and Ag- Ga exhibited round morphology with bright green fluorescence for cytoskeleton and clear nucleus similar to cells in the control group (Figure 10b), indicating healthy cell growth. The healthy sign of cells growing on Ag-Ga thin layer supported the finding that Ag-Ga suspension is cytocompatibility.

[0131] Cell migration is a process of cell movement from one area to another and is commonly used to access the wound healing.

[0047] In this study, the fibroblast (HFF) cells were cultured until they reached approximately 80% confluence. The cell surfaces were then deliberately scratched using a wound maker (as indicated by black dotted lines in Figure 10c). The remaining surfaces were incubated in growth medium, and cell migration across the scratched area was monitored in real-time using imaging techniques (IncuCyte SX5 live-cell analysis system).

[0132] To investigate the potential of the Ag-Ga suspensions to promote cell proliferation and migration, the remaining cell sheets were exposed to Ag-Ga conditioned medium (DMEM with 10% FBS). Microscopy images showed that the cells proliferated and migrated across the scratched area after 24 hours, with significant coverage of the area by 48 hours. In comparison, the positive control cells, treated with DMEM and 10% FBS, exhibited rapid cell migration and completely covered the scratched area by 48 hours. However, the cells treated with Ag-conditioned medium did not demonstrate any migration. These findings confirm that Ag-Ga suspension is compatible with cell proliferation and migration and has potential in biomedical applications.

[0133] Animal studies

[0134] In vivo antibacterial activity of Ag-Ga surface-coating implants

[0135] In recent years, biodegradable magnesium (Mg) metal has been a hot research topic.

[0048] The interest in this metal and its alloys stem for the fact that the elastic modulus of Mg is close to that of natural cortical bone.

[0049] Magnesium alloys are expected to be used as a bone conductive and biodegradable orthopedic implants and to enter clinical practice.

[0050] However, Mg and its alloys do notprovide protection against infection. In order to verify the applicability of the Ag-Ga particles, we coated them on Mg discs and evaluated their antibacterial activity in vivo (Figure 11a). For this experiment, we spray-coated Ga, Ag ion, and the Ag-Ga nano-amalgam suspension onto Mg discs. Next, the Mg (short for Mg discs), Ga@Mg (Ga coated group), Ag@Mg (Ag coated group), and Ag-Ga @ Mg (Ag-Ga coated group) discs were implanted subcutaneously in the lateral skin of the right hindlimb of each Sprague Dawley (SD) rat. Then, 1 x 106cfu of S. aureus or E. coli in 50 pL PBS were injected directly onto the disc surface to establish a subcutaneous bacterial infection (Figure lib). After 7 days, the rats were sacrificed, the implantation site was isolated and carefully opened to remove the discs. It was observed that some Magnesium discs have been absorbed. The internal surfaces were examined on day 7. As shown in Figure 11c, capsule formation surrounded the cavity where the implant was located. During the macroscopic examination of the bacteria-infected tissues, we observed the presence of obvious local abscesses specifically in the Mg group, and mild infections in the Ga@Mg and Ag@Mg groups, and the Ga@Mg group appeared to be more severe than the Ag@Mg group. Intriguingly, soft granulation tissue appeared after being implanted with Ag-Ga@Mg discs and there is no abscess, which indicated an antiinflammatory effect of the Ag-Ga coating (Figure 11c). Meanwhile, the changes in the blood test of the rats were monitored on the 14thday after surgery. A healthy trend of blood markers was measured for all groups, indicating that the inflammatory response was confined to the local area of infection and caused little alteration in the rats’ general health (Figure lid). Interestingly, although all data were within the normal range, the white blood cells, neutrophils, and lymphocytes in Ag-Ga@Mg group were the lowest among the 5 groups, indicating an inhibitory effect of the Ag-Ga coating on the systemic inflammatory response (Figure lid). After a two-week period, H&E staining was carried out on critical organs, including the heart, liver, spleen, lungs, and kidneys. Figure 12 shows no detectable damage to these organs, indicating that the material in question is histologically safe.

[0136] In addition, the skin at the implantation site was cut off and used for histopathology. Slices from the soft tissues at the surgical sites after 7 days of bacterial infection were processed by H&E staining, Masson staining, and Giemsa staining to evaluate the inflammatory infiltration, morphological changes, and bacterial residues of collected tissues, respectively. As demonstrated in Figure lie, significant necrosis, abscesses, and destruction of muscular tissue with infiltration of inflammatory cells, indicative of a typical soft tissue infection, were observed in both the control and Ga coating groups, whereas ameliorated signs of bacterial infection were discovered in the Ag coating group, which was consistent with the findings presented in Figure 11c. On the contrary, the combination of the Ag-Ga coating group significantly improved the outcomes of subcutaneous bacterial infection compared to other groups. In addition, the Immunohistochemical results indicated that the expression of inflammatory markers COX-2, INOS, and macrophage marker CD68 decreased in the Ag-Ga group relative to the other groups (Figure Ilf, g).

[0137] In vivo evaluation in an MRSA-induced rat femoral osteomyelitis model

[0138] Osteomyelitis is an infection caused by microorganisms and accompanied by an inflammatory process and bone destruction.

[0051] The use of implants is a major risk factors for the occurrence of osteomyelitis. Because implants significantly elevate the risk of osteomyelitis, prioritizing the prevention and treatment of peri-prosthetic joint infection (PJI) is crucial for managing the condition.

[0052] The main pathogenic bacteria of osteomyelitis are Staphylococcus aureus and coagulase-negative staphylococci (CoNS) such as Staphylococcus epidermidis and Staphylococcus lugdunensis.l53]In orthopedics, antibiotic resistance remains a challenge during treatment of osteomyelitis. About 50% of implant related infections are caused by Staphylococcus aureus,

[0054] especially MRSA.

[0139] The excellent biocompatibility of Ag-Ga coating prompted us to establish a MRSA-induced rat femoral osteomyelitis model in male Sprague Dawley rats using traditional Titanium rod to assess the antibacterial efficacy and bone regeneration ability of the material in vivo (Figure 13a). The total observation results of the rat femoral specimens are shown in Figure 13b, and the X-ray results of the femurs of different groups of rats are shown in Figure 13c. After injecting MRSA bacterial suspension into the femoral marrow cavity of rats for 2 weeks, cortical bone destruction, osteolysis, and periosteal reaction appeared in the femur of the rats, indicating the occurrence of osteomyelitis. X-ray evaluation showed that the Ti group of rats showed obvious signs of osteolysis and periosteal reaction. However, very few signs of osteolysis were observed in the Ag-Ga@Ti group (Figure 13c). Femurs were collected from rats at 14 days postoperatively and examined for osteolysis and tissue regeneration via micro-CT (Figure 13d). Micro-CT results revealed the presence of MRSA-induced cavitation in the femoral structures of the Ti, Ag@Ti, and Ga@Ti groups at 14 days after surgery. However, the Ag-Ga@Ti group exhibited superior bone repair compared to the other groups. Interestingly, the bone mineral density (BMD), the bone volume to total volume (BT / TV), trabecular number (Tb.N.), and trabecular thickness (Tb.Th) were much higher for the group treated with Ag-Ga@ Ti than for the other three groups, while the trabecular separation (Tb.Sp) was the lowest, implying the superior antimicrobial effect and osteogenic properties of Ag-Ga coating (Figure 13e and Figure 14).

[0140] H&E staining was performed to detect the degree of inflammation in the bone tissue surrounding the implants (Figure 15a). After a 2-week treatment period, the Ti and Ga@Ti groups exhibited a significant presence of inflammatory cells, including lymphocytes and neutrophils. In contrast, the Ag@Ti and Ag-Ga@Ti groups showed minimal infiltration of inflammatory cells. These observations were consistent with the results obtained by the Giemsa staining (Figure 15b). Additionally, Masson staining revealed a notable reduction in the inflammatory response and increased collagen deposition in the bone tissues treated with Ag-Ga@Ti (Figure 15c). To further investigate the immune microenvironment of infected bone tissue, we performed immunohistochemical staining of inflammatory markers. Notably, the Ag-Ga@Ti group exhibited the lowest expression levels of iNOS (Figure 15d, e), and COX-2 (Figure 15f, g), which are widely recognized biomarkers of inflammation.

[0055] This indicates adecrease in the inflammatory response within the Ag-Ga@Ti-treated tissues. By providing these comprehensive findings, we contribute to a better understanding of the anti-inflammatory effects of the Ag-Ga coating. These results highlight the potential of this new material as a promising treatment option for reducing inflammation in infected bone tissue.

[0141] To further investigate the ability of the Ag-Ga coating to promote bone regeneration, we conducted immunohistochemical staining of the femur to examine indicators associated with osteogenesis. The results, presented in Figure 15h-k, demonstrated a higher expression of Collagen I and TGF-J31 in the Ag-Ga@ Ti group, providing compelling evidence for the excellent osteogenic performance of the Ag-Ga coating. These findings support the notion that the Ag-Ga coating possesses desirable antibacterial and osteogenic properties, particularly in the context of deep tissue infections. Furthermore, the coating showed potential in countering MRSA, thereby reducing bone tissue erosion and facilitating bone tissue regeneration. Notably, its effectiveness in transparent cartilage regeneration was also noteworthy.

[0142] In vivo promotion of osteogenic ability in rat critical-size calvarial defect model

[0143] The implantation of a process that is particularly vital for addressing larger bone defects, it is vital for the biomaterial to facilitate bone regeneration. In this experiment, a critical-size rat skull defect model was used to assess the effectiveness of Ag-Ga coating-induced bone healing (Figure 16a).

[0056] Briefly, a skull defect with a diameter of 8 mm was generated in the parietal bone using a drill. The defect was centered around the sagittal suture line, specifically between lambda and the anterior chamber. Then the Mg discs sprayed with Ga, the Ag ion, and the Ag-Ga nano-amalgam suspensions were carefully inserted into the bone defect (Figure 16b). After 8 weeks, the recovery of skull defects in the different rat groups was evaluated by Micro-CT analysis. Figure 16c displayed the 3D reconstruction of Micro-CT images of rat skull defects at the 8-week mark. Notably, there were significant differences among the four groups: Micro-CT images of the bone defect showed that Ag-Ga coating significantly accelerated the formation of new bones after 6 weeks. Compared with the control group, the range of bone defects in the pure Mg discs decreased, but there is still a significant difference compared to the Ag-Ga coating group.

[0144] Then, histological analysis was conducted to analyze the tissue in the area of the skull defect. At 8 weeks after surgery, significant differences in differentiation were observed in the sagittal plane of the skull defect, as shown in Figure 16d. H&E staining revealed an increased amount of generated tissue in the Ag-Ga@Mg group compared to the other three groups (Figure 16d). Furthermore, Masson's trichrome staining demonstrated a substantial formation of bone-like matrix and collagen in the scaffold treated with Ag-Ga@Mg discs, while the other groups only showed a small number of collagen fibers (Figure 16e), which is consistent with the Micro-CT results. Additionally, immunohistochemical staining was performed on BMP-2, BMPR, and Runx-2 to further evaluate the effectiveness of Ag-Ga coating on bone regeneration. Immunohistochemical staining revealed a larger area of BMP-2, BMPR, and Runx-2positive cells in the Ag-Ga @ Mg group compared to the other three groups (Figure 16f). Moreover, the expression of BMP-2, BMPR, and Runx-2 was quantitatively analyzed using ImageJ software. Statistical analysis indicated that the Ag-Ga @ Mg group exhibited significantly higher expression levels of osteogenic factors compared to the Ga@Mg and Ag@Mg groups, which means the Ag-Ga coating significantly accelerated the formation of new bones (Figure 16g).

[0145] Conclusion

[0146] In summary, we developed a simple and controllable method for synthesizing Ag-Ga nanoamalgamated particles in the form of a suspension, which can be spray-coated onto variety of material surfaces to impart antibacterial properties. The method employs gallium liquid metal to reduce Ag ions in an aqueous solution (AgNOs) through galvanic replacement. The critical role of gallium liquid metal (GaLM) in the Ag-Ga nano-amalgamated particles is multifaceted and pivotal. GaLM not only serves as a carrier for silver nanocrystals through the galvanic replacement process, but also facilitates a controlled, slow-release mechanism for silver ions, which attenuates the pronounced toxicity associated with high concentrations of Ag ions. The Ag-Ga nano-amalgam suspension displayed a remarkable potential in creating surfaces that combat prevalent pathogenic bacteria, such as P. aeruginosa and S. aureus, achieving an efficacy nearly equivalent to that of silver ions at similar concentrations. Interestingly, GaLM mitigate the pronounced toxicity of Ag+, resulting in an Ag-Ga amalgam that is biocompatible with human fibroblast and keratinocyte cells. In contrast, these cells displayed a significant decrease in viability when exposed to Ag only coated surfaces. In vivo studies using a rat model further highlight the antibacterial potency of the Ag-Ga coatings, specifically against methicillin-resistant S. aureus and E. coli, when placed on implants such as titanium rods and magnesium discs. Moreover, the outcomes of the histological analyses conducted on rat models indicated that implants coated with silver-gallium demonstrated histologically to be non-toxic to tissue. Evaluations of the animal internal organs revealed that the normal functionality was maintained for two weeks subsequent to the transplantation procedure. Additionally, the Ag-Ga coating was associated with a prominent formation of bone-like matrix and collagen scaffolding, without eliciting an inflammatory response. The attributes of the Ag-Ga, as detailed in this study, underscore the potential of this new material as a prime candidate for generating antimicrobial and biocompatible coatings, making them suitable for prevention of infections with a range of medical materials and devices.

[0147] The following are examples of application of the compositions and methods described herein.

[0148] Example 1 - An antibacterial composition comprising silver-gallium (Ag-Ga) nanoamalgamated particles.

[0149] Example 2 - An antibacterial composition comprising silver-gallium (Ag-Ga) nanoamalgamated particles, synthesized through the galvanic deposition of silver nanocrystals in the oxidelayer on gallium liquid metal (GaLM).

[0150] Example 3 - The composition of examples 1 and 2, which includes copper, bismuth, and gold.

[0151] Example 4 - The composition of examples 1, 2 and 3 where polymers, oligomers, proteins, or other nanoparticles and ligands are incorporate within or on the surface of the nano-amalgamated particles.

[0152] Example 5 - The composition of examples 1 to 4, wherein the GaLM facilitates a controlled- release mechanism for silver and other metals, thereby improving biocompatibility.

[0153] Example 6 - The composition of examples 1 to 4, wherein the GaLM enhances the antibacterial properties of the other metal in the composition.

[0154] Example 7 - The composition of examples 1 to 4, wherein the GaLM facilitates reduction of inflammation and stimulates tissue growth.

[0155] Example 8 - A method for synthesizing particles as compositions of examples 1, 2 and 3, comprising the steps of using GaLM to facilitate the galvanic deposition of metal nanocrystals on oxide layer, where GaLM serves as carriers for other metals through the galvanic replacement process.

[0156] Example 9 - The method of example 8 further comprising a step of spray-coating Ag-Ga suspensions on devices and material surfaces for antibacterial purposes.

[0157] Example 10 - The composition of examples 1 to 4, wherein a stable coating is formed by methods such as spraying, dip coating or other coating technology.

[0158] Example 11 - The coating of example 10, wherein the coating is applied to the surface of medical device regardless of whether they are metallic, polymeric, ceramic or composite.

[0159] Example 12 - The coating of example 10, wherein the coating is applied to the surface of devices of complex shape.

[0160] Example 13 - The coating of example 10, wherein the coating has antibacterial properties.

[0161] Example 14 - The coating of example 10, wherein the coating has anti-inflammatory properties.

[0162] Example 15 - The coating of example 10, wherein the coating stimulates tissue regeneration.

[0163] Example 16 - The coating of example 10, wherein the coating simultaneously possesses all properties described in examples 12-14.REFERENCES

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[0225] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge.

[0226] It will be understood that the terms “comprise” and “include” and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification, and the claims that follow, is to be taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied.

[0227] In some cases, a single embodiment may, for succinctness and / or to assist in understanding the scope of the disclosure, combine multiple features. It is to be understood that in such a case, these multiple features may be provided separately (in separate embodiments), or in any other suitable combination. Alternatively, where separate features are described in separate embodiments, these separate features may be combined into a single embodiment unless otherwise stated or implied. This also applies to the claims which can be recombined in any combination. That is a claim may be amended to include a feature defined in any other claim. Further a phrase referring to “at least one of’ a list of items refers to any combination of those items, including single members. As an example, “at least one of: a, b, or c” is intended to cover: a, b, c, a-b, a-c, b-c, and a-b-c.

[0228] It will be appreciated by those skilled in the art that the disclosure is not restricted in its use to the particular application or applications described. Neither is the present disclosure restricted in its preferred embodiment with regard to the particular elements and / or features described or depicted herein. It will be appreciated that the disclosure is not limited to the embodiment or embodiments disclosed, butis capable of numerous rearrangements, modifications and substitutions without departing from the scope as set forth and defined by the following claims.

Claims

CLAIMS1. A composition of matter comprising silver-gallium (Ag-Ga) amalgamated particles.

2. The composition of matter of claim 1, comprising from about 0.01 at. % to about 70 at. % silver in the Ag-Ga amalgamed particles.

3. The composition of matter of claim 2, comprising about 1.5 at. %, about 3 at. %, about 4 at. % or about 6 at. % silver in Ag-Ga amalgamed particles.

4. The composition of matter of claim 2, comprising about 6 at. % silver in Ag-Ga amalgamed particles.

5. The composition of matter of any one of claims 1 to 4, further comprising a surfactant.

6. The composition of matter of claim 5, wherein the surfactant is a poloxamer.

7. The composition of matter of claim 6, wherein the poloxamer is a Pluronic™ surfactant.

8. The composition of matter of claim 7, wherein the Pluronic™ surfactant is Pluronic™ F-127.

9. The composition of matter of any one of claims 1 to 8, wherein the Ag-Ga amalgamed particles are nanoparticles.

10. The composition of matter of claim 9, wherein the average diameter of the Ag-Ga amalgamed nanoparticles is from about 1 nm to about 350 nm.

11. The composition of any one of claims 1 to 10, wherein the silver-gallium (Ag-Ga) amalgamated particles are formed by galvanic deposition of silver nanocrystals on an oxide layer of gallium liquid metal (GaLM).

12. A solution comprising the silver-gallium (Ag-Ga) amalgamated particles of any one of claims 1 to 11.

13. A coating comprising the silver-gallium (Ag-Ga) amalgamated particles of any one of claims 1 to 11.

14. A device comprising the coating of claim 13.

15. The device of claim 14, wherein the device is a medical device.

16. An antimicrobial composition comprising the silver-gallium (Ag-Ga) amalgamated particles ofany one of claims 1 to 11.

17. An antimicrobial coating comprising the silver-gallium (Ag-Ga) amalgamated particles of any one of claims 1 to 11.

18. The antimicrobial coating of claim 17, wherein the coating comprises from about 0.05 pg Ag / cm2to about 300 pg Ag / cm2.

19. The antimicrobial coating of either claim 17 or claim 18, wherein the coating comprises from about 0.10 pg Ga / cm2to about 300 mg Ga / cm2.

20. The antimicrobial coating of any one of claims 17 to 19, wherein the coating is antibacterial.

21. The antimicrobial coating of claim 20, wherein the coating achieves 99% elimination rates for S. aureus and P. aeruginosa at concentrations of 16 pg Ag / cm2and 176 pg Ga / cm2.

22. A device comprising the antimicrobial coating of any one of claims 17 to 21.

23. A process for producing silver-gallium (Ag-Ga) amalgamated particles, the process comprising mixing a solution comprising Ag+with a suspension comprising Ga particles under conditions to form silver-gallium (Ag-Ga) amalgamated particles.

24. The process of claim 23, comprising forming the suspension comprising Ga particles by subjecting a suspension of gallium in water to sonication.

25. The process of claim 24, wherein the sonication is for a period of from about 15 seconds to about 60 minutes.

26. The process of claim 25, wherein the sonication is for a period of about 25 minutes.

27. The process of any one of claims 24 to 26, wherein the suspension of gallium in water is cooled during the sonication.

28. The process of any one of claims 23 to 27, wherein the conditions to form silver-gallium (Ag-Ga) amalgamated particles comprise galvanic deposition of silver nanocrystals in the oxide layer on gallium liquid metal (GaLM).

29. The process of any one of claims 23 to 28, further comprising treating the silver-gallium (Ag-Ga) amalgamated particles with a surfactant.

30. The process of claim 29, wherein the surfactant is a poloxamer.

31. The process of claim 30, wherein the poloxamer is a Pluronic™ surfactant.

32. The process of claim 31, wherein the Pluronic™ surfactant is Pluronic™ F-127.

33. Silver-gallium (Ag-Ga) amalgamated particles produced by the process of any one of claims 23

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