INTRODUCTION

[0001] The present invention relates to nanoparticles comprising an inverse vulcanised sulfur polymer and a method of forming said nanoparticles. The invention also relates to a water filtration membrane comprising the nanoparticles, as well as sorbents comprising the nanoparticles. The invention also relates to the use of such nanoparticles in heavy metal remediation or extraction. The invention also relates to methods of removing heavy metals from fluids.

BACKGROUND OF THE INVENTION

[0002] Conventional vulcanisation has been known and used for over 170 years and has transformed the modern age - allowing greater industrialisation and mass transport. The vulcanisation of rubber is a multi-billion dollar industry worldwide, from vehicle tyres integral to modern transport to gaskets for complex machinery and even space flight. Conventional vulcanisation involves the use of small amounts of sulfur (typically no more than 0.5 to 3 wt%) to crosslink long chain organic polymers like natural rubber. As the polymers are usually solids, this requires complex and expensive machinery with a necessity for mechanical mixing (such as calendaring or screw extrusion).

[0003] In 2013 a new process of “Inverse Vulcanisation” was discovered (Nature Chemistry, 5, 518-524, 2013). This process uses small organic molecules to crosslink and stabilise polymeric sulfur (without this crosslinker, pure polymeric sulfur readily depolymerises to its monomer, Ss). This has allowed stable polymeric materials of up to 90 wt% sulfur to be produced. Unlike conventional vulcanisation, this process can be completed in the liquid phase, requiring no complex equipment.

[0004] Sulfur as an element has been known for more than a thousand years. ¹ From the First Industrial Revolution, the worldwide demand for elemental sulfur has soared massively, even leading to the Sulfur Crisis of 1840. ²³ However, in the modern world, an excessive supply of elemental sulfur is generated from the petrochemicals industry via the hydrodesulfurisation process, in order to decrease the emission of sulfur dioxide in the combustion of fossil fuels. ⁴ Therefore, alternative routes for the use of sulfur have been explored in recent decades, such as for concrete construction and lithium-sulfur batteries. ⁴⁵

[0005] Inverse vulcanisation, reported in 2013, gave a promising approach to use elemental sulfur in large amounts. ⁶ In this process, a solvent-free system, molten sulfur reacts with small organic molecules, normally divinylic monomers, to generate stable, high sulfur content polymers. The main theoretical mechanisms could be divided in two steps, namely, (1) generation of sulfur diradicals from homolytic cleavage of sulfur rings, and (2) reacting and capping of sulfur diradicals by C=C double bonds. 1 ,3-diisopropenylbenzene (DIB) was the first crosslinker to be explored in inverse vulcanisation, and the product, poly(S-DIB), was a chemically stable and processable copolymer, which could be used in cathodes for Li-S batteries. Subsequently to this discovery, more inverse vulcanisation crosslinkers have been studied, such as limonene, ⁷ dicyclopentadiene (DCPD), ⁸ diallyl disulfide, ^9-11 divinylbenzenes (DVB), ¹² perrilyl alcohol (PA), ¹³ ethylidene norbornene (ENB), ¹⁴ and ethylene glycol dimethacrylate (EGDMA). ^{15 16}

[0006] Meanwhile, a variety of corresponding applications, such as IR optics, ⁴ ’ ^{17 18} LiS batteries, ⁴ ’ ^{12 19} construction materials, ⁴ antimicrobial materials, ²⁰²¹ controlled-release fertilisers, ²² adhesives, ¹¹ and mercury capture, ^7-923-25 were discovered due to these new materials’ special properties. In many cases, to achieve a better performance in a given application, the morphology of sulfur polymers was studied as well. To demonstrate their shape-persistency or test their mechanical properties, sulfur polymers are often cured in silicone moulds. ⁶⁸ For Li-S batteries, sulfur polymers are normally ball milled into fine powder. ²⁶ In order to assess the optical properties, such as the transparency in the infrared region, inverse vulcanised polymers are often processed into thin films. ⁴

[0007] There have been even more efforts made in optimising the morphology of sulfur polymers to improve their performance in mercury adsorption. According to Pearson’s hard- soft-acid- base (HSAB) principle, sulfur is a “soft” Lewis base and mercury is a “soft” Lewis acid. Thus, it was found sulfur containing polymers had high affinity for mercury. In this application the morphology of inverse vulcanised polymers is crucial because the higher the specific surface area of materials, the larger contacted interface adsorbents can provide to the adsorbates. To this end, typical strategies involve coating polymers onto particles or substrates, ¹⁶ ’ ²⁴ ’ ^{25 27} electrospinning fibres blended with other polymers, ²⁸ templating by salt, ²⁹ or foaming the inverse vulcanised polymer with supercritical CO2 to generate porous structures. ³⁰ Almost all of these methods used auxiliary materials to support sulfur polymers and increase their surface area. Additionally, the efficiency of most of these strategies is low, a potential barrier for use on an industrial scale. Accordingly there is a need for a scalable, low cost sulfur-based polymeric material with a high potential for mercury capture.

[0008] The present invention was devised with the foregoing in mind.

SUMMARY OF THE INVENTION

[0009] In a first aspect, the invention provides nanoparticles comprising an inverse vulcanised sulfur polymer. [0010] In another aspect, the invention provides a method of forming nanoparticles comprising an inverse vulcanised sulfur polymer, the method comprising: a) providing a solution comprising an inverse vulcanised sulfur polymer dissolved in a first solvent; and b) contacting the solution with a second solvent, wherein the inverse vulcanised sulfur polymer is substantially insoluble in the second solvent; wherein contacting the solution with the second solvent results in the precipitation of nanoparticles comprising the inverse vulcanised sulfur polymer.

[0011] In another aspect, the invention provides an inverse vulcanised sulfur polymer as defined herein.

[0012] In another aspect, the invention provides a water filtration membrane comprising nanoparticles as defined in the first aspect of the invention.

[0013] In another aspect, the invention provides a sorbent comprising nanoparticles as defined in the first aspect of the invention.

[0014] In another aspect, the invention provides the use of nanoparticles as defined in the first aspect of the invention in heavy metal remediation.

[0015] In another aspect, the invention provides a method of removing heavy metals from a fluid, the method comprising; a) contacting the fluid with nanoparticles as defined in the first aspect of the invention; and b) separating the fluid from the nanoparticles as defined in the first aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Nanoparticles of the Present Invention

[0016] As described hereinbefore, an aspect of the invention provides nanoparticles comprising an inverse vulcanised sulfur polymer.

[0017] The inverse vulcanised sulfur polymer which the nanoparticles of the present invention comprise may be as defined anywhere herein.

[0018] The inverse vulcanised sulfur polymer may have a molecular weight (M _w ) of greater than 1000 Da. Suitably, the inverse vulcanised sulfur polymer has a molecular weight of from 1000 to 500,000 Da. More suitably, the inverse vulcanised sulfur polymer has a molecular weight of from 1000 to 50,000 Da.

[0019] The inverse vulcanised sulfur polymer may have a glass transition temperature (T _g ) of at least 10 °C. Preferably, the inverse vulcanised sulfur polymer may have a glass transition temperature of at least 30°C.

[0020] Suitably, the inverse vulcanised sulfur polymer has a glass transition temperature of from 10 to 200 °C. More suitably, the inverse vulcanised sulfur polymer has a glass transition temperature of from 30 to 75 °C.

[0021] The inverse vulcanised sulfur polymer may have a dispersion index (M _w /Mn) of from 1 to 10. Suitably, the inverse vulcanised sulfur polymer has a dispersion index (M _w /Mn) of from 1 to 5. Yet more suitably, the inverse vulcanised sulfur polymer has a dispersion index (Mw/Mn) of from 2 to 5.

[0022] The nanoparticles may have a particle size (a particle diameter) of from 1 to 5000 nm. Suitably, the nanoparticles have a particle size of from 50 to 3500 nm. More suitably, the nanoparticles have a particle size of from 100 to 1000 nm.

[0023] In the nanoparticles of the present invention, the inverse vulcanised sulfur polymer has a solubility of >1 mg/mL at 25°C in at least one polar aprotic solvent or non-polar solvent. More suitably, the inverse vulcanised sulfur polymer has a solubility of >25 mg/mL at 25°C in at least one polar aprotic solvent or non-polar solvent. More suitably, the inverse vulcanised sulfur polymer has a solubility of >50 mg/mL at 25°C in at least one polar aprotic solvent or non-polar solvent.

[0024] Suitably, the polar aprotic solvent or non-polar solvent may be selected from chloroform, tetra hydrofuran, toluene, acetone, hexane, carbon disulphide, dichloromethane, xylene, dimethylformamide and pyridine. Most suitably, the polar aprotic solvent or nonpolar solvent is chloroform.

[0025] Suitably, the inverse vulcanised sulfur polymer has a solubility of >1 mg/mL at 25°C in at least one of the following solvents: chloroform, tetrahydrofuran, toluene, acetone, hexane, carbon disulphide, dichloromethane, xylene, dimethylformamide and pyridine. More suitably, the inverse vulcanised sulfur polymer has a solubility of >1 mg/mL at 25°C in chloroform.

[0026] More suitably, the inverse vulcanised sulfur polymer has a solubility of >25 mg/ml at 25°C in at least one of the following solvents: chloroform, tetrahydrofuran, toluene, acetone, hexane, carbon disulphide, dichloromethane, xylene, dimethylformamide and pyridine. More suitably, the inverse vulcanised sulfur polymer has a solubility of >25 mg/mL at 25°C in chloroform. [0027] More suitably, the inverse vulcanised sulfur polymer has a solubility of >50 mg/ml at 25°C in at least one of the following solvents: chloroform, tetrahydrofuran, toluene, acetone, hexane, carbon disulphide, dichloromethane, xylene, dimethylformamide and pyridine. More suitably, the inverse vulcanised sulfur polymer has a solubility of >50 mg/mL at 25°C in chloroform.

[0028] Suitably, the inverse vulcanised sulfur polymer of the nanoparticles of the present invention is substantially insoluble (i.e. has a solubility of <1 mg/mL) at 25°C in at least one polar protic solvent or polar aprotic solvent.

[0029] Suitably, the inverse vulcanised sulfur polymer is substantially insoluble (i.e. has a solubility of <1 mg/mL) at 25°C in at least one of the following solvents: water, ethanol, methanol, acetone, propanol, isopropanol, ethyl acetate, diethyl ether and acetonitrile.

[0030] Suitably, the inverse vulcanised sulfur polymer is linear or has some degree of branching, but is not highly or fully crosslinked. A highly crosslinked polymer is understood to have a gel-phase as its major component, meaning that the inverse vulcanised sulfur polymer may not have the desired solubility properties to allow formation of the nanoparticles by the methods described herein. Suitably, the inverse vulcanised sulfur polymer comprises a gel fraction in an amount of less than 50 wt.%.

[0031] The particular solubility properties of the inverse vulcanised sulfur polymers defined herein allow the formation of nanoparticles without utilising surfactants. Suitably therefore, the nanoparticles of the first aspect of the invention do not comprise a surfactant.

[0032] The nanoparticles of the invention may comprise inverse vulcanised sulfur polymers having the solubility properties defined herein. However, it is also possible that once nanoparticles have been formed using the method of the invention, the polymers within the nanoparticles are further reacted to create a more crosslinked network (for example by heating). Thus, it is not essential that the final nanoparticles comprise inverse vulcanised sulfur polymers which have the solubility profiles described herein.

[0033] The inverse vulcanised sulfur polymer is suitably formed from the reaction between elemental sulfur (Ss) with one or more organic co-monomers, wherein the organic comonomers) comprises at least one reactive carbon-carbon double bond (i.e., one or more reactive carbon-carbon double bonds). Suitably, the one or more organic co-monomers have a molecular weight of less than 1000 g/mol, e.g. less than 500 g/mol e.g. from 40 to 1000 g/mol or from 40 to 300 g/mol.

[0034] Suitably, the inverse vulcanised sulfur polymer is formed from the reaction between sulfur and one or more co-monomers selected from: perillyl alcohol, dicyclopentadiene, di- isopropenyl benzene, di-vinyl benzene, styrene, terpinolene, limonene, myrcene, farnasene, farnasol, eugenol allyl ether or methyl methacrylate.

[0035] Suitably, the inverse vulcanised sulfur polymer is selected from one or more of: a) a binary co-polymer of sulfur-perillyl alcohol, sulfur-terpinolene, sulfur-di- isopropenyl benzene, sulfur-dicyclopentadiene or sulfur-styrene; and/or b) ternary co-polymers of sulfur-limonene-dicyclopentadiene, sulfur-perillyl alcoholdicyclopentadiene or sulfur-styrene-dicyclopentadiene.

[0036] The inverse vulcanised sulfur polymer may comprise sulfur in an amount of at least 30 wt% (by weight of the polymer). Suitably, the inverse vulcanised sulfur polymer comprises sulfur in an amount of at least 40 wt%. More suitably, the inverse vulcanised sulfur polymer comprises sulfur in an amount of at least 50 wt%.

[0037] The inverse vulcanised sulfur polymer may comprise sulfur in an amount of from 30 to 80 wt%. Yet even more suitably, the inverse vulcanised sulfur polymer comprises sulfur in an amount of from 40 to 70 wt%, or from 40 to 60 wt.%.

[0038] Suitably, the weight ratio of sulfur to the one or more co-monomers in the inverse vulcanised sulfur polymer is from 0.5: 1 to 4: 1 , more suitably from 0.75: 1 to 2: 1 or from 0.9: 1 to 1.1 :1 (e.g. 1 :1).

[0039] Suitably, the inverse vulcanised sulfur polymer is a binary co-polymer of sulfur and a single co-monomer, e.g. sulfur-perillyl alcohol, sulfur-terpinolene, sulfur-di-isopropenyl benzene, sulfur-dicyclopentadiene or sulfur-styrene. Suitably, the weight ratio of sulfur to the co-monomer is as defined herein, e.g. from 0.5: 1 to 4: 1 , more suitably from 0.75: 1 to 2: 1 or from 0.9:1 to 1.1 :1 (e.g. 1 :1).

[0040] Suitably, the inverse vulcanised sulfur polymer is a ternary co-polymer of sulfur- limonene-dicyclopentadiene, sulfur-perillyl alcohol-dicyclopentadiene or sulfur-styrene- dicyclopentadiene. suitably, the weight ratio of sulfur to the total weight of the co-monomers is as defined herein, e.g. from 0.5: 1 to 4: 1 , more suitably from 0.75: 1 to 2: 1 or most suitably from 0.9:1 to 1.1 :1 (e.g. 1 :1).

[0041] Suitably, the inverse vulcanised sulfur polymer comprises: a. sulfur; b. a first co-monomer selected from limonene, perillyl alcohol and styrene; c. a second co-monomer which is dicyclopentadiene; wherein the weight ratio of sulfur to the co-monomers is from 0.5: 1 to 4: 1 (e.g. 0.75: 1 to 2:1); and the first co-monomer makes up from 60 to 100 wt.% of the total weight of the comonomers.

[0042] Suitably therefore the weight ratio of the first co-monomer to the second comonomer is from 0.6:0.4 to 1 :0 (e.g. 0.6:0.4, 0.7:0.3, 0.8:0.2, 0.9:0.1 , 1 :0).

[0043] In an embodiment, the ternary co-polymer comprises: sulfur; a first named co-monomer; and a second named co-monomer; wherein the weight ratio of sulfur: first co-monomer:second co-monomer is:

0.75-1.5 : 0.6-1 : 0-0.4; and the first named co-monomer is selected from limonene, perillyl alcohol or styrene, and the second named co-monomer is dicyclopentadiene.

[0044] More suitably, the ternary co-polymer comprises: sulfur, a first named co-monomer and a second named co-monomer in a weight ratio of 0.9 - 1.1 : 0.6 - 1 : 0 - 0.4.

[0045] Most suitably, the ternary co-polymer comprises sulfur, a first named co-monomer and a second named co-monomer in a weight ratio of: a. 1 :1 :0 (sulfur : first co-monomer : second co-monomer); b. 1 :0.9:0.1 ; c. 1 :0.8:0.2; d. 1 :0.7:0.3; or e. 1 :0.6:0.4.

[0046] Most suitably, the inverse vulcanised sulfur polymer is a ternary co-polymer selected from: sulfur-limonene-dicyclopentadiene in a weight ratio of 1 :0.9:0.1 (sulfur : limonene : dicyclopentadiene); sulfur-perillyl alcohol-dicyclopentadiene in a weight ratio of 1 :0.9:0.1 (sulfur : perillyl alcohol : dicyclopentadiene); or sulfur-styrene-dicyclopentadiene having a weight ratio of 1 :0.9:0.1 (sulfur : styrene : dicyclopentadiene).

[0047] The inverse vulcanised sulfur polymer present in the nanoparticles of the present invention, or utilised in the method of forming the nanoparticles of the present invention may be any of the inverse vulcanised sulfur polymers disclosed herein or in the specific embodiments below.

[0048] In an embodiment, the inverse vulcanised sulfur polymer has a glass transition temperature of from 10 to 200 °C, and has a molecular weight of from 1000 to 500,000 Da.

[0049] In an embodiment, the inverse vulcanised sulfur polymer has a glass transition temperature of from 10 to 200 °C and a dispersion index (M _w /Mn) of from 1 to 10.

[0050] In an embodiment, the inverse vulcanised sulfur polymer has a molecular weight of from 1000 to 500,000 Da, and a dispersion index (M _w /Mn) of from 1 to 10.

[0051] In an embodiment, the inverse vulcanised sulfur polymer has a glass transition temperature of from 10 to 200 °C, and has a molecular weight of from 1000 to 500,000 Da, and a dispersion index (M _w /Mn) of from 1 to 10.

Methods of Forming Nanoparticles of the Present Invention

[0052] The present inventors have found that nanoparticles comprising inverse vulcanised sulfur polymers can be formed by contacting a solution of a dissolved inverse vulcanised sulfur polymer with a second solvent (an “antisolvent”) in which the inverse vulcanised sulfur polymer is substantially insoluble. The inverse vulcanised sulfur polymer will precipitate from the solution when the solution is contacted with the second solvent.

[0053] Another aspect of the invention provides a method of forming nanoparticles comprising an inverse vulcanised sulfur polymer, the method comprising: a) providing a solution comprising an inverse vulcanised sulfur polymer dissolved in a first solvent; and b) contacting the solution with a second solvent, wherein the inverse vulcanised sulfur polymer is substantially insoluble in the second solvent; wherein contacting the solution with the second solvent results in the precipitation of nanoparticles comprising the inverse vulcanised sulfur polymer.

[0054] The inverse vulcanised sulfur polymer may be any of the inverse vulcanised sulfur polymers described herein.

[0055] The inverse vulcanised sulfur polymer may have a molecular weight of greater than 1000 Da. Suitably, the inverse vulcanised sulfur polymer has a molecular weight of from 1000 to 500,000 Da. More suitably, the inverse vulcanised sulfur polymer has a molecular weight of from 1000 to 50,000 Da.

[0056] The inverse vulcanised sulfur polymer may have a glass transition temperature of at least 10 °C. Suitably, the inverse vulcanised sulfur polymer has a glass transition temperature of from 10 to 200 °C. More suitably, the inverse vulcanised sulfur polymer has a glass transition temperature of from 30 to 50 °C.

[0057] Suitably, the inverse vulcanised sulfur polymer has a dispersion index (M _w /Mn) of from 1 to 10. More suitably, the inverse vulcanised sulfur polymer has a dispersion index (M _w /Mn) of from 1 to 5. Yet more suitably, the inverse vulcanised sulfur polymer has a dispersion index (M _w /Mn) of from 2 to 5.

[0058] The inverse vulcanised sulfur polymer may be linear or include some branching, rather than being highly or fully crosslinked. A highly crosslinked inverse vulcanised sulfur polymer is understood to have a gel-phase as its major component, meaning that it will not have the desired solubility properties. Suitably, the inverse vulcanised sulfur polymer is not so highly crosslinked that it is substantially insoluble in all of the solvents disclosed herein, for example chloroform.

[0059] Suitably, the inverse vulcanised sulfur polymer has a solubility of >1 mg/mL at 25°C in the first solvent. More suitably, the inverse vulcanised sulfur polymer has a solubility of >25 mg/mL at 25°C in the first solvent. More suitably, the inverse vulcanised sulfur polymer has a solubility of >50 mg/mL at 25°C in the first solvent.

[0060] The first solvent may be any solvent in which the inverse vulcanised sulfur polymer can dissolve. Suitably, the first solvent may be a polar aprotic solvent or a non-polar solvent. Suitably, the first solvent is selected from chloroform, tetrahydrofuran, toluene, acetone, hexane, carbon disulphide, dichloromethane, xylene, dimethylformamide and pyridine. More suitably, the first solvent is chloroform.

[0061] The second solvent may be any solvent in which the inverse vulcanised sulfur polymer has a solubility of less than 1 mg/mL.

[0062] The second solvent may be a polar protic solvent or a polar aprotic solvent.

[0063] Suitably, the second solvent is selected from water, ethanol, methanol, acetone, propanol, isopropanol, ethyl acetate, diethyl ether and acetonitrile. More suitably, the second solvent is ethanol.

[0064] Suitably, the second solvent is at least partly miscible with the first solvent. More suitably, the second solvent is miscible with the first solvent.

[0065] Suitably, the second solvent is added dropwise to the first solvent. [0066] Suitably, the second solvent is added to the first solvent with stirring.

[0067] In the method of forming nanoparticles of the present invention, it may be that neither the first nor second solvent comprise a surfactant. Avoiding the use of a surfactant in the method of the present invention advantageously avoids contaminating the surface of the formed nanoparticles, allowing greater opportunity for heavy metal binding.

[0068] In the method of forming nanoparticles of the present invention, the inverse vulcanised sulfur polymer may be any of the inverse vulcanised polymers described herein.

[0069] Prior to step a), the method may suitably further comprise the step of preparing an inverse vulcanised sulfur polymer by reacting elemental sulfur with one or more organic comonomers, wherein the organic co-monomer(s) comprises at least one reactive carboncarbon double bonds (i.e. , one or more reactive carbon-carbon double bonds). The formed inverse vulcanised sulfur polymer will suitably be dissolved in the first solvent.

[0070] Suitably, the step of preparing an inverse vulcanised sulfur polymer is achieved by reacting elemental sulfurwith one or more organic co-monomers at a temperature of at least 120°C, more suitably at least 140°C or most suitably at least 160°C

[0071] Suitably, the one or more co-monomers are selected from perillyl alcohol, dicyclopentadiene, di-isopropenyl benzene, di-vinyl benzene, styrene, terpinolene, limonene, myrcene, farnasene, farnasol, eugenol allyl ether or methyl methacrylate.

[0072] Suitably, the one or more co-monomers are selected from perillyl alcohol, di- isopropenyl benzene, di-vinyl benzene, styrene, terpinolene, limonene, myrcene, farnasene, farnasol, eugenol allyl ether or methyl methacrylate.

[0073] Suitably, the one or more co-monomers are selected from perillyl alcohol, geraniol, dicyclopentadiene, di-isopropenyl benzene, di-vinyl benzene, styrene, terpinolene, limonene, myrcene, farnasene, farnasol, eugenol allyl ether or methyl methacrylate.

[0074] Suitably, the inverse vulcanised sulfur polymer may be formed using a combination of co-monomers to obtain an inverse vulcanised sulfur polymer with the required properties defined herein.

[0075] Suitably, the co-monomer may only be partially reacted with the sulfur, (partly cured) such that the sulfur polymer is not fully crosslinked. This allows the required properties of the inverse vulcanised sulfur polymer (e.g. glass transition temperature, solubility and/or molecular weight) to be obtained. Such an approach is common in the field of carbon-based polymers depending on the end use of the polymer.

[0076] Suitably, the inverse vulcanised sulfur polymer is selected from: a) binary co-polymers of sulfur-perillyl alcohol, sulfur-terpinolene, sulfur-di- isopropenyl benzene, sulfur-dicyclopentadiene or sulfur-styrene; and/or b) ternary co-polymers of sulfur-limonene-dicyclopentadiene, sulfur-perillyl alcoholdicyclopentadiene or sulfur-styrene-dicyclopentadiene.

[0077] Suitably, the inverse vulcanised sulfur polymer is a binary co-polymer of sulfur- perillyl alcohol, sulfur-terpinolene, sulfur-di-isopropenyl benzene, sulfur-dicyclopentadiene or sulfur-styrene as described herein.

[0078] Suitably, the inverse vulcanised sulfur polymer is a ternary co-polymer of sulfur- limonene-dicyclopentadiene, sulfur-perillyl alcohol-dicyclopentadiene or sulfur-styrene- dicyclopentadiene as described herein.

[0079] The inventors have provided a method which allows the formation of stable, shape persistent, nanoparticles comprising an inverse vulcanised sulfur polymer. The inverse vulcanised sulfur polymer used in the method of the invention should have the solubility profiles and suitably one or more of the other properties described herein to allow successful formation of the nanoparticles. However, once the nanoparticles have been formed, further crosslinking of any unreacted C=C double bonds in the nanoparticles may be performed.

[0080] Suitably therefore, the method of the invention may further comprise the step of further crosslinking the inverse vulcanised sulfur polymer within the nanoparticles. The further crosslinking may be achieved by heating the nanoparticles to crosslink unreacted C=C double bonds within the nanoparticles. The nanoparticles may be heated to a temperature of at least 120°C, more suitably at least 140°C or most suitably at least 160°C.

[0081] Nanoparticles which have undergone a further crosslinking step following formation may be highly crosslinked. Thus, nanoparticles of the present invention may be substantially insoluble in the solvents described herein.

[0082] In another aspect of the present invention, there is provided nanoparticles obtained by, obtainable by, or directly obtained by the method of forming nanoparticles defined herein.

Inverse Vulcanised Sulfur Polymers of the Present Invention

[0083] The inventors have found that a particular combination of properties allows certain inverse vulcanised sulfur polymers to be utilised in an antisolvent precipitation method to form nanoparticles. For example, a suitable inverse vulcanised sulfur polymer must be soluble in the first solvent and substantially insoluble in the second solvent (antisolvent).

[0084] Thus, in another aspect, there is provided an inverse vulcanised sulfur polymer as defined anywhere herein or in the specific embodiments below. [0085] In an embodiment, the inverse vulcanised sulfur polymer has a glass transition temperature of from 10 to 200 °C, and has a molecular weight of from 1000 to 500,000 Da.

[0086] In an embodiment, the inverse vulcanised sulfur polymer has a glass transition temperature of at least 30 °C, and has a molecular weight of from 1000 to 500,000 Da.

[0087] In an embodiment, the inverse vulcanised sulfur polymer has a glass transition temperature of from 10 to 200 °C and a dispersion index (M _w /M _n ) of from 1 to 10.

[0088] In an embodiment, the inverse vulcanised sulfur polymer has a glass transition temperature of at least 30 °C, and a dispersion index (M _w /M _n ) of from 1 to 10.

[0089] In an embodiment, the inverse vulcanised sulfur polymer has a molecular weight of from 1000 to 500,000 Da, and a dispersion index (M _w /M _n ) of from 1 to 10.

[0090] In an embodiment, the inverse vulcanised sulfur polymer has a glass transition temperature of from 10 to 200 °C, has a molecular weight of from 1000 to 500,000 Da, and a dispersion index (Mw/Mn ) of from 1 to 10.

[0091] In an embodiment, the inverse vulcanised sulfur polymer has a glass transition temperature of at least 30 °C, a molecular weight of from 1000 to 500,000 Da, and a dispersion index (Mw/Mn ) of from 1 to 10. Suitably, the inverse vulcanised sulfur polymer has a solubility of >1 mg/mL at 25°C in one of the solvents described herein, e.g. chloroform.

[0092] The inverse vulcanised sulfur polymer present in the nanoparticles of the present invention, or utilised in the method of forming the nanoparticles of the present invention may be any of the inverse vulcanised sulfur polymers disclosed herein or in the specific embodiments above.

Applications and Uses

Water filtration membrane and sorbents

[0093] Another aspect of the invention provides a water filtration membrane comprising nanoparticles as defined herein.

[0094] In the water filtration membranes of the present invention, the nanoparticles may be present on a support. Suitably, the nanoparticles are supported on a porous membrane. More suitably, the nanoparticles are supported on a porous membrane selected from a polytetrafluoroethylene (PTFE) membrane or a nylon membrane.

[0095] Another aspect of the invention provides a sorbent comprising nanoparticles as defined herein.

[0096] In the sorbent of the present invention, the nanoparticles of the present invention may be present on a support. Suitably, the nanoparticles are supported on a porous material (e.g., a macro-porous material). The porous material may comprise an inorganic material selected from silica, an organosilicate, alumina, zeolite, titania and/or mica. Suitably, the porous material comprises silica, e.g., a silica bead. More suitably, the porous material comprises silica bead.

[0097] Alternatively, the porous material may comprise a fibre or a polymer. Suitably, the porous material comprises a filter paper, spun polypropylene fibres, spun nylon fibres and/or cotton fibres.

[0098] The porous materials or membranes will suitably contain pores in the macropore (>50 nm) range, provided that the nanoparticles can fit within the pores. Macropores are likely to be beneficial as transport pores.

[0099] The inventors have found that the nanoparticles of the present invention are suitable commercial candidates for heavy metal remediation, with their preparation both simple and scalable.

[00100] Furthermore, the inventors have advantageously found that the nanoparticles can be applied as highly selective sorbents in mercury removal from a range of samples ranging from extremely low mercury concentration (ppb level) to high mercury concentration (ppm level). Moreover, the inventors have also demonstrated these nanoparticles to be effective in mercury filter membranes.

[00101] Another aspect of the invention provides a use of nanoparticles as defined herein in heavy metal remediation.

[00102] Suitably, the heavy metal remediation is mercury, gold, platinum or lead remediation. More suitably, the heavy metal remediation is mercury remediation. Accordingly, the invention provides the use of nanoparticles as defined herein in mercury remediation. The use may be in the remediation of organomercury compounds, such as methyl mercury chloride and/or mercury salts such as mercury chloride.

[00103] Another aspect of the invention provides a use of nanoparticles as defined herein in the extraction of precious metals. Suitably, the extraction of precious metals is gold extraction.

[00104] Another aspect of the invention provides a method of removing heavy metals from a fluid, the method comprising; a) contacting the fluid with nanoparticles as defined herein; and b) separating the fluid from the nanoparticles as defined herein.

[00105] Suitably, the fluid comprises one or more of heavy metals, a heavy metal salt or a heavy metal compound. The concentration of heavy metals, including heavy metal salts and heavy metal compounds, in the fluid will be reduced after the nanoparticles are separated from the fluid.

[00106] The method may be a method of heavy metal remediation. The method may be a method of precious metal extraction.

[00107] The nanoparticles may be comprised within a mercury filter membrane as defined herein.

[00108] Suitably, the fluid is an aqueous mixture. The aqueous mixture may be a solution comprising a heavy metal, a heavy metal salt or a heavy metal compound.

[00109] The present invention has particular application in the removal of mercury from the environment. The aqueous mixture may comprise soil from the environment that is contaminated with mercury. Thus, the aqueous mixture may be a liquid comprising mercury, a mercury compound or a mercury salt. The aqueous mixture may be a mercury solution comprising mercury salt e.g. mercury chloride. The aqueous mixture may comprise an organomercury compound, e.g. methyl mercury chloride.

[00110] The nanoparticles of the present invention also find application in the removal of gold from the environment i.e. in gold extraction. Thus, the aqueous mixture may be a gold solution. Thus, the aqueous mixture may be a liquid comprising a gold salt such as gold chloride, gold fluoride, gold bromide, gold thiosulfate, gold cyanide, gold thiourea or gold sulfate.

[00111] The nanoparticles of the present invention may also be applied to Li-S batteries. Thus, there is provided a lithium-sulfur (Li-S) battery comprising nanoparticles as defined herein.

[00112] The nanoparticles of the present invention may also find use in catalysis. Thus, there is provided a catalytic composition comprising nanoparticles as defined herein.

Antibacterial and drug delivery applications

[00113] Another aspect of the invention provides a use of the nanoparticles as defined herein as antibacterial agents and/or drug delivery systems. The nanoparticles may be suitable as antibacterial agents in wound dressings, catheter coatings and/or antimicrobial surface coatings.

[00114] Accordingly, an aspect of the invention provides a use of nanoparticles as defined herein in wound dressings, catheter coatings, antimicrobial surface coatings and/or drug delivery systems. Definitions

[00115] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[00116] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[00117] The term “Inverse vulcanised sulfur polymer” or “sulfur polymer” for short, are taken to mean a polymer formed from elemental sulfur, which comprise polymeric chains of sulfide bonds, crosslinked by small molecule hydrocarbons. Thus, inverse vulcanisation is the reaction of elemental sulfur with a small molecule “crosslinker” or “co-monomer”. An example is the reaction with of elemental sulfur with diisopropenyl benzene:

[00118] The inverse vulcanised sulfur polymer therefore comprises polymeric polysulfide chains “crosslinked” to one another via the small molecule co-monomer. The inverse vulcanised sulfur polymer will therefore comprise polysulfide bonds to the co-monomer. The term “polysulfide bonds” will be understood to mean bonds of C-S-S-C or higher sulfur rank

(C-Sn-C where n >2), rather than only thioether bonds, such as C-S-C.

[00119] A conventionally vulcanised polymer on the other hand comprises numerous repeating units of a carbon-carbon backbone, crosslinked by sulfide bonds. Such polymers may be crosslinked by sulfur in the conventional vulcanisation process. However, the predominant component of such polymers is carbon and the sulfur content is much lower than in an inverse vulcanised sulfur polymer. Thus, conventional vulcanisation is the reaction of elemental sulfur with a polymer. An example is the vulcanisation of poly(isoprene): c ret ss- linke d po l (s ssp rene)

[00120] The term “a small molecule crosslinker” or “small molecule co-monomer” refers to a compound with a molecular weight of less than 1000 g/mol, e.g. from 40 to 1000 g/mol from 40 to 1000 g/mol or from 40 to 300 g/mol.

[00121] In the context of the present invention, the term “nanoparticle” means a particle having a particle size (diameter) of from 1 to 5000 nm.

[00122] It is preferred that the inverse vulcanised sulfur polymers used in the method of the present invention are linear or have some degree of branching so as to have the desired solubility. Suitably, the degree of crosslinking in the inverse vulcanised sulfur polymers is such that the polymer has one or more of the desired properties defined herein (e.g. solubility, T _g , molecular weight and/or dispersion index M _w /M _n ).

[00123] If the co-monomer used to form the inverse vulcanised sulfur polymer comprises multiple carbon-carbon double bonds, then the inverse vulcanised sulfur polymers used in the method of the invention will typically comprise some unreacted carbon-carbon double bonds. It may be that over 50% of the C=C double bonds remain unreacted during the antisolvent precipitation process. Once nanoparticles are formed however, the remaining unreacted bonds may be further reacted with elemental sulfur to increase the amount of crosslinking within the nanoparticles.

[00124] The term “linear” in the context of polymers generally (not just those of inverse vulcanised sulfur polymers) will be understood to mean that the polymer structure repeats along a continuous line.

[00125] The term “branched” in the context of polymers generally (not just those of inverse vulcanised sulfur polymers) will be understood to mean that the polymer structure forks and/or forms a T-junction. [00126] The term “fully crosslinked” or “highly crosslinked” in the context of polymers generally (not just those of inverse vulcanised sulfur polymers) will be understood to mean that all (or substantially all) possible locations for crosslinking have been utilised, i.e. the formed polymer comprises no reactive double bonds, or very few reactive double bonds. A fully or highly crosslinked inverse vulcanised polymer have a high gel content and will be substantially insoluble in all of the solvents described herein, for example chloroform, and therefore not be suitable for use in the method of the present invention.

[00127] The degree of crosslinking in the inverse vulcanised sulfur polymer can be assessed by melt rheology to determine if the structure is linear. If the polymer is crosslinked, then its molten state will not be observed in a temperature ramp test. After the T _g , the rubbery plateau area will extend up to higher temperatures until the polymer starts to decompose.

[00128] In the context of the present invention, the dispersion index (or polydispersity index, PDI) is used in two ways, one being related to the molecular weight of the inverse vulcanised sulfur polymers per se, and the other being related to the size of the nanoparticles comprising the inverse vulcanised sulfur polymers. When used in relation to molecular weight of the inverse vulcanised sulfur polymers perse, the term “dispersion index” is used and is the value for M _w /M _n (weight average molecular weight (M _w ) divided by the number average molecular weight (M _n )). The term polydispersity index (PDI) is used when referring the size distribution of the nanoparticles comprising the inverse vulcanised sulfur polymers. The PDI values are calculated by the software used in the dynamic light scattering equipment, and this value is presented as a percentage.

[00129] The dispersion index (M _w /M _n ) is also an indicator of the degree of crosslinking, with a higher value indicating more crosslinking. The degree of crosslinking may also be confirmed by NMR spectroscopy to identify the amount of unreacted carbon-carbon double bonds in the co-monomer.

[00130] If the polymer gel fraction exceeds 50 wt.% of the total polymer weight, then the polymer is not expected to be suitable for use in the method of the present invention. Suitably, the inverse vulcanised polymer comprises less than 50 wt.% gel fraction.

[00131] A skilled person will be able to adjust the reaction parameters to ensure that the inverse vulcanised polymer does not become fully crosslinked, i.e. by managing the extent of the curing. Such an approach is common in the vulcanisation of carbon based polymers, where the degree of crosslinking, i.e. curing (vulcanisation), is managed so as to obtain the desired properties in the final product. For example, the extent of the crosslinking when dicyclopentadiene is used as a co-monomer may be managed by reacting sulfur with only the more reactive cyclohexane C=C double bond, rather than on both of the double bonds. Managing the extent of the crosslinking in this way helps to obtain the desired solubility properties.

[00132] The term “reactive” in the context of carbon-carbon double bonds will be understood to mean that the carbon-carbon double bond can undergo one or more transformations. The carbon-carbon double bonds may be non-aromatic carbon-carbon double bonds. Suitably therefore the organic co-monomer used to form the inverse vulcanised sulfur polymer may be a vinyl compound. The vinyl compound may include one or more allyl groups (i.e., a substituent with the structural formula H2C=CH-CH2R, where R is the rest of the molecule). Suitable vinyl compounds also include substituted vinyl compounds, for example vinyl compounds optionally substituted with halogens, oxygen, acetates, acrylates and phosphates.

[00133] The term “substantially insoluble” will be understood to mean that the inverse vulcanised sulfur polymer has a solubility of <1 mg/mL at 25°C in the second solvent.

[00134] The term “aqueous mixture” refers to any mixture of substances which comprises at least 10 wt% water. It may comprise at least 30 wt% water. It may comprise at least 50 wt% water and preferably comprises at least 80 wt% water, e.g., at least 90 wt% water. The mixture may be a solution, a suspension, an emulsion or a mixture thereof. Typically, the aqueous mixture will be an aqueous solution in which one or more solutes are dissolved in water. This does not exclude the possibility that there might be particulate matter, droplets or micelles suspended in the solution.

BRIEF DESCRIPTION OF THE DRAWINGS

[00135] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, which are described below:

Figure 1 : a) Offset DSC traces for polysulfides with varied ratio of PA to DCPD. b) Tg trending with the change of DCPD/PA ratio.

Figure 2: Solubility study (spots + line) of polysulfides. Bar chart indicated the component ratio of designed reactant of SPD-50,Y,Z.

Figure 3: Correlation function of SPD-50, 45, 05-10 (top) and SPD-50, 45, 05-50 (bottom).

Figure 4: Correlation function of SPD-50, 45, 05-100 (top) and SPD-50, 45, 05-250 (bottom).

Figure 5: SEM images of SPD-50, 45, 05-10 (top), SPD-50, 45, 05-50 (middle) and SPD- 50, 45, 05-100 (bottom). Figure 6: Offset DSC traces for polysulfide nanoparticles, SPD-50, 50, 00-250, and bulk polysulfide SPD-50, 50, 00, polysulfide nanoparticles, SPD-50, 40, 10-250, and bulk polysulfide SPD-50, 40, 10.

Figure 7: GPC trace for polysulfides precursors, SPD-50, 45, 05, and polysulfides nanoparticles, SPD-50, 45, 05-250.

Figure 8: Correlation functions of SPD-50, 50, 00-250 (top), SDIB-50, 50-250 (middle) and SDCPD-50, 50-250 (bottom).

Figure 9: Mercury filter prototype produced by commercial syringe filter (a Nylon 0.45 pm syringe filter) with synthesized sulfur nanoparticles, SPD-50, 25, 05-250.

Figure 10: Selectivity test of mercury filter prototypes using mixed ion solution, simulating waste-water. More than 90 % mercury was selectively removed by mercury filter prototypes.

Figure 11 : Schematic for general inverse vulcanisation, and co-monomers, e.g. DCPD and perillyl alcohol. Also shown is a schematic for antisolvent precipitation and application of polysulfide nanoparticles in mercury capture.

Figure 12: a) size distribution of polysulfide nanoparticles characterised by DLS. b) and c) SEM images of SPD-50, 45, 05-10, showing polysulfide nanoparticles mostly uniform and spherical, as the example shown in c). d) SEM image of SPD-50, 45, 05-250, indicating although nanoparticles formed through antisolvent precipitation, the shape of the particles are irregular.

Figure 13: a) ¹ H NMR spectra of polysulfide nanoparticles, SPD-50, 45, 05-250, and bulk polysulfide, SPD-50, 45, 05 dissolved in CDCh. Vinylic protons of DCPD and perillyl alcohol were assigned and highlighted by blue band. The new generated peaks, highlighted by the red band, are assigned as H-C-S positions b) Offset DSC traces for polysulfide nanoparticles, SPD-50, 45, 05-250, and bulk polysulfide SPD-50, 45, 05.

Figure 14: a) Size distribution of polysulfide nanoparticles characterised by DLS. b) SEM image of SPD-50, 50, 00-250. c) SEM image of SDIB-50, 50-250. d) SEM image of SDCPD- 50, 50-250.

Figure 15: a) SEM image of polysulfide nanoparticles supported on the commercial 0.45 pm PTFE membrane, showing polysulfide nanoparticles and PTFE nanofibres, b) prototype of the inverse vulcanised sulfur polymer supported membrane for mercury removal.

Figure 16: a) Static mercury uptake test of various polysulfide nanoparticles with HgCl2 and CHsHgCl. b) Selectivity test of polysulfide nanoparticles using mixed ion solution, simulating waste-water. Mercury could be removed totally and selectively. Concentration of specific ions in the CRM is: Cr, 331 ppb, Mn, 1134 ppb, Ni 1215, ppb, Co, 876 ppb, As, 248 ppb, Se, 129 ppb, Cd, 434 ppb, Hg, 9.7 ppb, Pb, 243 ppb.

Figure 17: a) S. aureus growth curve in the presence of S50-PA nanoparticles over 24 h in nutrient-rich LB medium, b) P. aeruginosa growth curve in the presence of S50-PA nanoparticles after 5 h of incubation in nutrient-rich LB medium, c) Absorbance at 600 nm after staining with crystal violet after 24 and 48 h of incubation at 37 °C with S. aureus (USA300) and P. aeruginosa (PAO1). d) Cell viability (%) of HepG2 after treatment with S50- PA nanoparticles.

Figure 18: Graph summarising the % growth of S. aureus relative to a positive control, in the presence of S50-PA nanoparticles at various concentrations during a 24 h incubation period. ***p < 0.001 , ****p < 0.0001 compared to control.

Figure 19: S. aureus growth curve in the presence of S50-Ger nanoparticles after 5 h incubation in nutrient-rich LB medium.

Figure 20: S. aureus growth curve in the presence of S70-PA nanoparticles after 5 h incubation in nutrient-rich LB medium.

Figure 21 : Graph summarising the % growth of P. aeruginosa relative to a positive control, in the presence of S50-PA nanoparticles at various concentrations during a 24 h incubation period. *p < 0.05, ****p < 0.0001 , NS denotes a value that is not statistically significant compared to the control.

Figure 22: P. aeruginosa growth curve in the presence of S50-Ger nanoparticles after 5 h incubation in nutrient-rich LB medium.

Figure 23: % growth of B9 compared to untreated culture in the presence of tobramycin (512-1 pg/mL), and a combination of tobramycin (512-1 pg/mL) and S50-PA nanoparticles (128 pg/mL).

EXAMPLES - part A

[00136] Antisolvent precipitation is a versatile method to prepare micro- and nanoparticles, widely used in food and pharmaceutical industries. ^31-35 This technique presents several advantages, such as low cost, easy processing and scaling up, potential solvent recovery and few opportunities for contamination. However, the formation of micro- and nanoparticles of sulfur polymers has not been demonstrated. Reported here for the first time, is the production of sulfur polymer nanoparticles by antisolvent precipitation, yielding nanomaterials with high potential for mercury capture. The inventors have established that a balance of particular properties of the sulfur polymer is required to successfully produce nanoparticles by antisolvent precipitation.

Materials

[00137] Sulfur (Ss, sublimed powder, reagent grade, > 99.5 %, Brenntag UK & Ireland), dicyclopentadiene (DCPD > 96.0 %, Sigma-Aldrich), (S)-(— )-perillyl alcohol (> 95 %, Sigma Aldrich) mercury (II) chloride (ACS, 99.5% MIN, Alfa Aesar UK), Chloroform-d (99.8 atom % D, Sigma Aldrich). Chloroform (Stabilized with amylene, Fisher Scientific), Ethanol (> 99.5 %, Sigma-Aldrich), 1 ,3-diisopropenyl benzene (> 97.0 %(GC), TCI), Trace Metal Certified Reference Material (QC3132-500ML, Lot LRAC5492, Sigma-Aldrich). All chemical precursors were used as received without any further purification. Deionized water was used in filtration and washing steps of the resultant materials.

Synthesis of inverse vulcanised sulfur polymers

Poly( Sulfur-Perillyl Alcohol-Dicyclopentadiene)

[00138] Reactants (sulfur and co-monomers, 10 g in total, specific ratio for different samples are indicated by X, Y, Z in sample name, SPD-X,Y,Z, presenting the mass percentage of sulfur, PA, and DCPD, respectively) were mixed in 40 mL volume glass vials. The mixture was stirred at 175 °C in aluminium blocks and stirred (800 rpm) by magnetic stirrer bars. The reaction time depended on the ratio of reactants. When the reaction had changed to thick dark brown liquid, the prepolymer was transferred into a silicone mould and moved into an oven at 140 °C for 18h.

Poly (sulfur-diisopropenyl benzene)

[00139] Reactants (sulfur and co-monomers, 1 :1 in weight ratio, 10 g in total) were mixed in 40 mL volume glass vials. The mixture was stirred at 175 °C in aluminium blocks and stirred by magnetic stirrer bars. The reaction time depended on the ratio of reactants. When the reaction had changed to thick dark brown liquid, the prepolymer was transferred into a silicone mould and moved into an oven at 140 °C for 18h. The sample generated was named SDIB-50,50.

Partly cured poly(Sulfur-dicyclopentadiene)

[00140] Reactants (sulfur and dicyclopentadiene, 1 :1 in weight ratio, 10 g in total) were mixed in 40 mL volume glass vials. The mixture was stirred at 175 °C in aluminium blocks and stirred by magnetic stirrer bars. The reaction time depended on the ratio of reactants. When the reaction had changed to thick dark brown liquid, the prepolymer was transferred into a silicone mould and moved into an oven at 140 °C for 3h. The sample generated was named SDCPD-50,50. SDCPD-50,50 and SPD-50,00,50 were different samples, as SDCPD-50,50 is partly cured and SPD-50,00,50 is fully cured, though the reactant and the ratio of reactant were exactly same.

Preparation of sulfur polymer nanoparticles

[00141] 500 mg of specific sulfur polymer were dissolved in 10 mL of chloroform (CHCh) (solvent) to generate a 50 mg mL ^-1 polymer solution, with all insoluble parts filtered out. 10 pL, 50 pL, 100 pL, and 250 pL of solution was added to 10 mL ethanol (anti-solvent) dropwise with stirring (500 rpm) at room temperature to precipitate nanoparticles. Samples were denoted as SPD-X,Y,Z-A, where A is the volume of polymer solution in microlitres. Generated nanoparticles were filtered under vacuum using a PTFE membrane (< 0.2 pm pore size), then, dried in the vacuum oven at room temperature overnight.

Fabrication of sulfur polymer supported membrane

[00142] 50 mg sulfur polymer nanoparticles were added into 50 mL ethanol and well dispersed by sonication. When prepared, the mixture was a cloudy light yellow or light grey suspension, depending on sulfur polymer precursors. 20 mL resultant suspension was filtered by a PTFE membrane (< 0.2 pm pore size) using glass vacuum filtration, generating sulfur polymer supported membrane.

Preparation of mercury filter prototype

[00143] Sulfur polymer suspension was prepared same as mentioned above. 10 mL resultant suspension was added into a 12 mL syringe connected with a commercial syringe filter, followed by drying in the vacuum oven at room temperature overnight.

Mercury uptake

Static mercury uptake test

[00144] 10 ppm HgCl2 and 10 ppm CHsHgCI solution were prepared by dilution of a 1000 ppm stock solution. The required mass of nanoparticles was placed into a centrifuge tube with 10 mL solution. Nanoparticles were dispersed well by sonication for 1 min. Each solution was left to agitate on a tube roller at 60 rpm for 24h. The subsequent mixtures were separated by use of a Nylon 0.45 pm syringe filter. The filtrate was then stabilized with 1 mL HNOs(aq), and analysed via ICP-OES, using an Agilent 5110 ICP-OES spectrometer.

Selectivity study

[00145] 10 mL Trace Metal Certified reference material (CRM) were pipetted into 14 mL glass sample vials. To each tube 10 mg of nanoparticles was added. Blank sample was prepared by adding no samples. Each solution was left to agitate on a tube roller at 60 rpm for 1 h. The subsequent mixtures were separated by use of a Nylon 0.45 pm syringe filter. The filtrate was then stabilized with 1 mL HNOs(aq). Samples were then analysed via inductively coupled plasma mass spectrometry (ICP-MS), using Perkin Elmer Nexion 2000 ICP-MS with a Meinhard nebuliser and cyclonic spray chamber.

Mercury removal by mercury filter prototype

[00146] 10 mL Trace Metal Certified reference material (CRM) were pipetted into 12 mL syringe with a mercury filter prototype. Blank sample was prepared by adding CRM into 12 mL syringe with a syringe filter. All solutions were filtered manually within 10s. The filtrate was then stabilized with 1 mL HNOs(aq). Samples were then analysed via inductively coupled plasma mass spectrometry (ICP-MS), using Perkin Elmer Nexion 2000 ICP-MS with a Meinhard nebuliser and cyclonic spray chamber.

Characterization

Dynamic Light Scattering (DLS)

[00147] The size of polysulfide nanoparticles was detected by Litesizer™ 500. Nanoparticles were dispersed into ethanol by sonication, generating a nanoparticle dispersion with concentration of 0.2 mg mL ^-1 . 1.5 mL of each samples were pipetted into a 2.5 mL standard disposable cuvette and analysed at room temperature (25 °C). All measurements were carried with a fixed backscattering angle of 175° using automated setting of a maximum of 60 runs.

Scanning electron microscopy (SEM)

[00148] Morphology images of the composite were achieved using a Hitachi S-4800 cold field emission scanning electron microscope (FE-SEM). Samples were prepared by adhering nanoparticle powder to an adhesive carbon tab and subsequently sputter coating with chromium (sputter time: 25s; current 120 mA; distance: -100 mm; specimens rotated during coating).

Nuclear magnetic resonance (NMR) [00149] The reactions were monitored by solution NMR in deuterated chloroform, using a Bruker Avance DRX (400 MHz) spectrometer.

Gel permeation chromatography (GPC)

[00150] The molecular weight of the soluble fraction of the polymers was determined by GPC using a Viscotek system comprising a GPCmax (degasser, eluent and sample delivery system), and a TDA302 detector array, using THF as eluent.

Elemental analysis (CH NS)

[00151] Elemental analysis samples were submitted to the University of Liverpool, Chemistry Department Micro-Analysis service and tested by an Elementar Vario Micro Cube.

Results

[00152] Initially, sulfur polymers, poly(Sulfur-Perillyl Alcohol-Dicyclopentadiene) (poly(SPD)), were synthesized through inverse vulcanisation methods (Figure 11 , see above for experimental details). Ternary systems or copolymer blends are commonly used in inverse vulcanisation to increase the reactivity of monomers or adjust the physicochemical properties or final products. ⁹³⁶ Fully cured poly(S-Dicyclopentadiene) (poly(S-DCPD)) is a fully crosslinked sulfur polymer, which is insoluble in solvents. ⁸ Poly(S-Perillyl Alcohol) (poly(S-PA)) usually has a low glass transition temperature (T _g ) and low molecular weight, preventing shape persistence at ambient temperature. Therefore, SPD was synthesized to generate a soluble sulfur polymer but with higher T _g and molecular weight whilst maintaining solubility. SPD products are denoted as SPD-X,Y,Z, where X, Y, Z indicate the mass percentage of sulfur, PA, and DCPD, respectively. Compared with SPD-50,50,00 (T _g = 31 °C), as shown in Figure 1 , SPD-50,45,05 and SPD-50,40,10 had higher T _g values, 39 and 47 °C respectively, both of which are above room temperature. The solubility of the terpolymer decreases significantly as more DCPD, a co-monomer, is added, especially over 30 wt.% DCPD (Figure 2). SPD-50,45,05 was further investigated as this gave a sufficient increase to M _w and T _g , while still maintaining high solubility

[00153] 500 mg of SPD-50,45,05 (solute) was initially dissolved in 10 mL of chloroform (CHC ) (solvent) to generate a 50 mg mL ^-1 polymer solution. 10 pL, 50 pL, 100 pL, and 250 pL of solution was added to 10 mL ethanol (anti-solvent) dropwise with stirring (500 rpm) at room temperature to precipitate nanoparticles (solubility study shown in Table 1).

Table 1. Solubility of representative sulfur polymers

Note: 500 mg of polymer powder was placed in 10 mL solvent and agitated overnight. Soluble = Solubility >50 mg/mL, Partly Soluble = 50 mg/mL > Solubility > 1 mg/mL, Insoluble = Solubility < 1 mg/mL.

[00154] No surfactant was involved in the synthesis to keep the processing facile and to avoid contaminating the surface of nanoparticles, which are used as sorbents for heavy metals. Samples were denoted as SPD-50,45,05-A, where A is the volume of polymer solution in pL. From DLS results, as shown in Figure 12a, it can be found that sulfur nanoparticles were successfully prepared with z-average diameter from -100 nm to 1000 nm. The correlograms of these particles (Figure 3) show a smooth decay as expected for particles of this size range which are suitable for analysis by DLS. As more polymer solution was added, a second size range, from -2000 nm to 3500 nm, can be observed. The increase in average hydrodynamic diameter of these particles with increasing polymer content is evident from a slightly slower decay in the correlation function (Figure 4). SPD-50, 45, 05-10 formed the most uniform nanoparticles, which have narrow size distributions (polydispersity index, PDI, 23 %), with hydrodynamic diameter of 486 nm. As the solvent to anti-solvent ratio increased, the distribution of particle size became much broader, and the hydrodynamic diameter increased to 590 nm in the case of SPD-50, 45, 05-50, indicating an increase in particle size as well. This result aligned with reported studies that particle size can be reduced by increasing the anti-solvent-to-solvent ratio. ³¹ In SPD-50, 45, 05-100 and SPD- 50, 45, 05-250, both distributions were broader and aggregation was more prevalent. The morphology of nanoparticles prepared under different conditions was observed through scanning electron microscopy (SEM). Similar to the conclusion from DLS testing, it could be found that the shape of SPD-50, 45, 05-10 (Figures 5 (top), 12b and 12c) is more uniform than others (Figures 5 (middle and bottom) and 12d) , although all particles analysed by SEM appeared smaller size than that shown in DLS, which is to be expected as DLS measures the hydrodynamic radius while SEM images are obtained on the dried particles. SEM also revealed that the shape of SPD-50,45,05-10 is more uniform and spherical than that of SPD- 50,45,05-250, which also had other irregular shapes, such as elongated spheroids and aggregates. However, considering that the application of sulfur polymer nanoparticles is to uptake mercury, non-uniform shapes and aggregation are acceptable as their increased surface area to volume ratio increases the productivity. Additionally, large diameters I larger particle size assists retrieval of the particles by sedimentation, filtration, or centrifugation. Therefore SPD-50, 45, 05-250 was selected as a representative sample for further characterisation and application test.

[00155] From ¹ H NMR spectroscopy of SPD-50, 45, 05, as shown in Figure 13a, it was found that C=C double bonds of both perillyl alcohol (vinylic protons: from 4.0 ppm to 4.7 ppm) and DCPD (vinylic protons: from 5.5 ppm to 5.9 ppm) fully reacted. The broad peaks from 3.6 to 3.7 ppm were assigned as the protons on the generated S-C-H, further confirming the reaction of sulfur and C=C double. Compared with SPD-50, 45, 05, SPD-50, 45, 05-250 has negligible differences in ¹ H NMR spectroscopy, indicating the chemical structure of polymer has no obvious change before and after antisolvent precipitation. Elemental Analysis for both SPD-50, 45, 05 and SPD-50, 45, 05-250 was performed, showing a slight difference before and after treatment, as shown in Table 2.

Table 2. Calculated element contents and detected element contents

[00156] It is theorised that this is because the dissolution of the polymer extracts lower molecular weight polymers which then do not precipitate when added to the anti-solvent, thus shifting the elemental composition. In the comparison of DSC results, as shown in Figure 13b, nanoparticles, SPD-50, 45, 05-250, have higher T _g = 63 °C than that (T _g = 33 °C) of bulk materials, SPD-50,45,05. Similar results that nanoparticles showed higher T _g than their corresponding precursors are observed in other samples (Figure 6). It is theorised that the reason for these results is that small molecules and oligomers, acting as a plasticiser and suppressing the T _g in the pristine polymer, could be dissolved in antisolvent and washed out, leaving only relatively high molecular weight polymers to generate nanoparticles. This is supported by GPC results, as shown in Figure 7; the M _n and M _w of SPD-50,45,05 was 455 and 1242, respectively, and M _w /M _n = 2.734, while after antisolvent precipitation, M _n and M _w of SPD-50, 45, 05-250 was 669 and 1281 , respectively, and M _w /M _n = 1.916. Similar results were achieved from GPC results of SPD505000 (M _n = 608 and M _w = 809, M _w /M _n = 1.33)/SPD-50, 50, 00-250 (M _n = 724 and M _w = 907, M _w /M _n = 1.25) and SPD-50, 40, 10 (M _n = 713 and M _w = 2676, M _w /M _n = 3.76)/SPD-50,40, 10-250 (M _n = 1207 and M _w = 2906, M _w /M _n = 2.407). These indicate that increasing the ratio of DCPD increased the molecular weight of poly(SPD), aligning with the results from DSC testing, and that the average molecular weight of all poly(SPD) increased. The distribution also become more uniform, post antisolvent precipitation. Higher molecular weights and T _g values may make it possible for the sulfur polymer nanoparticles to be applied as mercury sorbents at room temperature, owing to the increased hardness and shape persistency. By the same method as before, other inverse vulcanised polymers, such as poly (sulfur-diisopropenyl benzene) (poly(S-DIB)), poly(S-PA), and partly cured poly(S-DCPD), were synthesized into nanoparticles, SDIB-50, 50-250, SPD-50, 50, 00-250, and SDCPD-50, 50-250, correspondingly, as shown in Figure 14 and the DLS correlograms can be seen in Figure 8. From both DLS (Figure 14a) and SEM results (Figure 14c), it could be found that SDIB-50, 50-250 had the most uniform size (PDI, 7.3 %) and spherical shape, and the smallest particle diameters (hydrodynamic diameter, 167 nm). However, nanoparticles at this size are not easily collected by filtration, compared to larger particles. The morphologies of SDCPD-50, 50-250 and SPD-50, 50, 00-250 were very similar, containing both spheres and other irregular shapes. However, SDCPD-50, 50-250 showed large amounts of aggregation, which speculatively is due to the same reason for the observed aggregation in SPD-50, 45, 05-250. Thus, the morphology of sulfur polymer nanoparticles is highly dependent on the precursors and the solvent-antisolvent system. Owing to relatively large particle sizes and aggregation, simple prototypes of mercury filter membranes could be prepared from sulfur polymer nanoparticles, by supporting them on commercial 0.45 pm PTFE membranes, as shown in Figure 15 and Figure 9.

[00157] Elemental sulfur, metal sulfides, and polysulfides are commonly used as mercury sorbents, because of the interaction between a “soft” Lewis base, sulfur; and a “soft” Lewis acid, mercury. Mercury uptake also benefits from porosity and high specific surface area. Therefore, various structures and shapes of polysulfides have previously been exploited. Nano-sized polysulfides demonstrated good performance and high efficiency in mercury capture, however, previously reported sulfur containing nanomaterials required other auxiliary materials, such as a porogen or support materials. ¹⁶ ’ ²⁴ ’ ^28-30 SPD-50, 45, 05-250, as well as other nanoparticles were tested as mercury sorbents for aqueous solutions. Initially, the required mass of nanoparticles was weighed into a centrifuge tube, followed by dispensing of 10 mL of aqueous HgCl2 solution of a required concentration. From the results, as shown in Figure 16a and Table 3, almost all aqueous mercury was removed from a 10 ppm HgCl2 solution by using 10 mg polysulfide nanoparticles, and more than 95% of mercury was removed from 10 ppm HgCh solution by using 5 mg polysulfide nanoparticles.

Table 3. Static mercury uptake results of polysulfide nanoparticles

[00158] Additionally, polysulfide nanoparticles were also applied in capturing methylmercury chloride from aqueous solution. From Figure 16a and Table 3, it could be found that the removal of methylmercury chloride is not as effective as HgCl2, however, all polysulfide nanoparticles could uptake more than 80% mercury from 10 ppm aqueous solution. These results out-performed a commercial mercury adsorbent, activated carbon, in similar initial concentration mercury solution (as shown in Table 4). ³⁹⁴⁰

Table 4. Comparison of different adsorbents for Hg ²⁺ uptake.

[00159] More specifically, in comparison with directly related bulk polysulfide materials made of the same components, (Table 4), polysulfide nanoparticles demonstrated significant improvement in mercury uptake. Taking poly(S-DCPD) as an example, the mercury capacity of SDCPD-50, 50-250 in static testing (Co = 10 ppm) reached 19.1 mg g ^-1 , much higher than that of the previously reported S-DCPD (0.1 mg g ^-1 , Co = 2 ppm), ⁸ saturation capacity of salt templated of S-DCPD (2.27 mg g ^-1 ), ²⁹ and saturation capacity of S-DCPD coated silica gel (5 mg g ^-1 ). ²⁷ Similarly, SPD-50, 50, 00-250 has much higher mercury capacity (19.5 mg g ^-1 , Co = 10 ppm) than its counterpart bulk materials (0.05 mg g' ¹ , Co = 2.5 ppm). ¹³ Not only did they perform better than the same component polysulfide in previously reported formulations for mercury uptake, these novel nanoparticles are also comparable positively to polysulfides synthesized by other crosslinkers, such as poly (S-r- Canola) (1.81 mg g ^-1 ), ³⁸ poly (S-r-Rice Bran) (1.92 mg g ^-1 ), ³⁸ poly(S-r-Castor) (2.01 mg g' ¹ ), ³⁸ and poly(sulfur-GOB-DCPD) (1.60 mg g ^-1 Co = 20 ppm). ⁹ The relatively simple preparation processing (with no auxiliary materials) and higher proportion of active component are a significant advantage of the inverse vulcanised polymer nanoparticles.

[00160] The efficiency of the sorbent to remove mercury in more realistic conditions was studied. ⁴¹ Mercury concentration in groundwater and surface water is less than 0.5 ppb, however, it could be up to 5.5 ppb in volcanically active locations. According to an exposure study, the World Health Organisation (WHO) suggested that 6 ppb of inorganic mercury in daily drinking water is a maximum guideline value for an adult. Therefore, lower mercury concentration solutions with competing metal ions were applied to simulate contaminated waste-water. All sulfur polymer nanoparticles were demonstrated to have high efficiency and high selectivity for mercury removal, as all mercury (9.70 ppb, 10 mL) was selectively removed from mixed metal ion solutions in 1 h, as shown in Figure 16b and Table 5.

Table 5. Selectivity test of polysulfide nanoparticles from mixed ion solution

Note: -’ means under detectable level of ICP-MS’ [00161] The mercury filter prototype produced by SPD-50, 45, 05-250 supported on commercial PTFE membrane filter, was tested upon a mixed ion solution. Compared with a blank membrane filter, which reduced the original mercury concentration from 7.52 ppb to 7.15 ppb, the mercury filter prototype performed with high efficiency and high selectivity for instantaneous filtration of mercury, which removed almost 90% of the mercury, 7.52 ppb to 0.78 ppb (average of three tests), a result more relevant in industry applicable conditions (shown in Figure 10 and Table 6).

Table 6. Selectivity test of mercury filter prototype from mixed ion solution

EXAMPLES - part B

Materials

[00162] Ground sulfur sublimed powder reagent grade >99.5% was obtained from Brenntag U.K. and Ireland. (S)-(— )-Perillyl alcohol food grade >95%, geraniol food grade >97%, Luria- Bertani broth (Miller), LB agar and phosphate buffered saline (PBS), crystal violet, cell proliferation kit I (MTT), and lead(ll) acetate test strips were purchased from Sigma-Aldrich. Eagle’s minimum essential medium (EMEM) was purchased from ATCC. Methicillin- resistant S. aureus strain USA300 and P. aeruginosa strains PAO1 and B9 were cultured from frozen stocks stored at the University of Liverpool.

Characterisation

Differential Scanning Calorimetry (DSC)

[00163] DSC was performed using a TA Instruments Q200 DSC, programmed using a heat/cool/heat method for three cycles by heating to 150 °C, cooling to -80 °C, and reramping to 150 °C. The heating/cooling rate was set to 10 °C/min. The second heating curve was analysed and used to determine the glass transition temperature. Fourier transformed infrared spectroscopy (FT-IR)

[00164] FT-IR data was obtained on a Bruker TENSOR 27 FT-IR, between 400 and 4000 cm ^-1 using an attenuated total reflectance accessory.

Nuclear magnetic resonance (NMR)

[00165] NMR samples were analysed using a Bruker Advance DRX (400 MHz) spectrometer using deuterated chloroform as the solvent; all experiments were carried out at room temperature.

Dynamic light scattering (DLS)

[00166] DLS measurements were obtained at 25 °C on a Malvern Instruments Ltd. Zetasizer Nano Series Nano-ZS spectrometer using the automatic attenuator and measurement position settings. The z-average diameter was measured using 1 cm path length disposable cuvettes. Nanoparticles were dispersed at a range of concentrations to determine a size independent of the concentration.

Scanning electron microscopy (SEM)

[00167] SEM was performed using a Hitachi S-4800 cold-field emission scanning electron microscope. The nanoparticle dispersions were dropped onto silicon wafer chips, which were subsequently mounted onto SEM stubs using conductive silver paint. For imaging cells after treatment with nanoparticles, diluted S. aureus cultures supplemented with nanoparticles, or water as a control, were incubated for 5 h. The cultures were pelleted by centrifugation and washed with PBS. The pellets were incubated overnight in glutardialdehyde and then dispersed and spun down in a series of ethanol dilutions (50, 70, 90, 95, and 100% v/v). The pellets were redispersed in ethanol, and the resulting dispersions were dropped onto silicon wafers. Prior to imaging, samples were coated with gold using a current of 120 mA for 15 s to give approximately 15 nm gold coatings using a Quorum S1505 ES sputter coater.

Absorbance measurements for the MTT assay

[00168] Obtained using a BMG Labtech FLUOstar Omega microplate reader using 96-well plates.

Synthesis of High Sulfur Content Polymers

[00169] Polymerization was carried out in 40 mL glass vials placed in aluminium heating blocks. Sulfur/cross-linker weight ratios of 50 and 70 wt % sulfur were used, with the total reaction scale maintained at 10 g. All reactions begun by allowing the sulfur to melt at 135 °C before adding the organic cross-linker under stirring. The reaction temperature was increased to 175 °C. Molten prepolymer was poured into silicone moulds followed by curing overnight in an oven at 140 °C. The cured polymers were allowed to cool and were ground into powders using a pestle and mortar.

Preparation of Polymeric Nanoparticles

Emulsion-Solvent Evaporation Method

[00170] Nine millilitres of aqueous surfactant solution (varying concentrations) was added to a 14 mL glass vial. One millilitre of S50-Ger in chloroform (5 mg/mL, 50 wt % S) was added to the vial and immediately sonicated for 40 s. The vial was equipped with a 15 mm x 6 mm magnetic stirrer bar, and the resulting cream-colored emulsion was allowed to stir at 600 rpm and at room temperature overnight or until the chloroform had evaporated.

Nanoprecipitation Method

[00171] Nine millilitres of aqueous surfactant solution (10 mg/mL) or distilled water was added to a 14 mL glass vial and allowed to stir at room temperature at 600 rpm. One millilitre of S-Ger or S-PA in tetrahydrofuran (THF) (varying concentrations, 50 and 70 wt % S) was added to the aqueous solution dropwise with continued stirring at 600 rpm. Once 1 mL of the polymer dissolved in THF was added to the vial, the solution was continued to stir at room temperature at 600 rpm overnight or until THF had evaporated fully.

Bacteria Preparation, Storage, and Enumeration

[00172] Glycerol stocks of S. aureus strain USA300 and P. aeruginosa strain PAO1 were stored at -80 °C for long-term storage. For experimental use, frozen glycerol stocks of S. aureus and P. aeruginosa were defrosted and spread onto LB agar plates, which were then incubated overnight at 37 °C. Bacterial cultures were prepared by swabbing one colony into 10 mL of LB broth, followed by overnight incubation at 37 °C. Colony forming units (CFUs) were enumerated by serially diluting the cultures in PBS onto LB agar, using the Miles and Misra method. CFU/cm ² and CFU/mL were calculated using the following equation:

CFU/mL=(No. of colonies x total dilution factor) /volume of culture plated in mL

Viable Bacterial Cell Enumeration Assay [00173] S. aureus LISA300 and P. aeruginosa PAO1 were used to evaluate the antibacterial efficiency of the high sulfur content polymeric nanoparticles. Blank samples were prepared by dropping 1 mL of THF into 9 mL of water followed by overnight stirring (600 rpm) at room temperature to allow for the THF to evaporate. Overnight cultured bacteria prepared in LB broth were diluted to 10 ⁵ CFU/mL (OD600 = 0.001) in LB or M9 media. Nine hundred microlitres of diluted bacterial solution was added to 2 mL vials along with 100 pL of nanoparticles dispersed in water (final concentration of nanoparticles: 14, 27, 55, 220, and 440 pg/mL) or blank. The samples were incubated for 5 h at 37 °C on a roller. Viable cells were enumerated after serial dilution of the solution in PBS onto LB agar, using the Miles and Misra method at 0, 10, 30, 60, 90, 120, and 300 min.

Determination of the Minimum Inhibitory Concentration

[00174] Minimum inhibitory concentrations of sulfur nanoparticles were assessed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, (16) for an incubation period of 24 h against S. aureus strain USA300 and P. aeruginosa strain PAO1 , in LB medium. An initial OD600 of 0.1 (~5 x 10 ⁵ CFU/mL) was used for the cell cultures prior to incubation. Nanoparticles were tested at 2-fold dilutions spanning a concentration range of 0.5-512 pg/mL. The OD600 was measured using a FLUOstar Omega microplate reader. The same method was used to investigate the antibacterial activity of the nanoparticles, combined with tobramycin, against the extensively drug resistant P. aeruginosa strain B9, isolated from an acute respiratory infection in Thailand.

Bio film Staining Assay

[00175] S. aureus USA300 and P. aeruginosa PAO1 were used to evaluate biofilm formation in the presence of high sulfur content polymeric nanoparticles. Blank solutions were prepared by dropping 1 mL of THF into 9 mL of water followed by overnight stirring (600 rpm) at room temperature to allow for the THF to evaporate. Overnight cultured bacteria prepared in LB broth was diluted to 10 ⁵ CFU/mL (OD600 = 0.001). Nine hundred microlitres of diluted culture and 100 pL of nanoparticles dispersed in water (final concentrations of 220 and 440 pg/mL) or blank solution were added to separate wells of a 24-well plate. The well plate was incubated statically at 37 °C for 24 and 48 h. After incubation, the solutions from the well plate were discarded and it was rinsed with 1 mL of PBS, which was then discarded and the plate was allowed to dry. The dried wells were stained with 1 mL 0.25% crystal violet for 30 min. The dye was discarded, and the well plate was thoroughly rinsed with water and allowed to dry. One millilitre of ethanol was added to each well in order to solubilize any remaining dye. The absorbance at 600 nm was measured using ethanol as a blank.

Cell Culture

[00176] HepG2 cells were maintained in Eagle’s minimum essential medium (EMEM) cell culture medium (ATCC) supplemented with 10% fetal bovine serum (FBS). Cells were maintained in a 5% CO2 incubator at 37 °C.

Cell Viability Assay

[00177] Cell viability was evaluated using the MTT assay. HepG2 cells were seeded at a concentration of 5 x 10 ⁴ cells/well in 100 pL culture medium and incubated (5% CO2, 37 °C) for 48 h or until approximately 80% confluent. Culture media was removed and replaced with 90 pL of culture medium and 10 pL of nanoparticles (final concentration of nanoparticles: 14, 27, 55, 220, and 440 pg/mL) or blank solution. The cells in the presence of nanoparticles/blank were incubated for 24 h (5% CO2, 37 °C). After 24 h of incubation, the media containing nanoparticles/blank was removed and replaced with 100 pL of culture medium and 10 pL of the MTT labelling reagent (final concentration of 0.5 mg/mL). The microplate was incubated for a further 4 h (5% CO2, 37 °C). One hundred microliters of the solubilizing buffer was added to each well, and the microplate was allowed to stand overnight in the incubator. The solubilization of the purple formazan crystals was measured at an absorbance wavelength of 595 nm.

Statistical Analysis

[00178] Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Tukeys post hoc test. Differences were deemed as statistically significant if a value of p < 0.05 was obtained.

Results

[00179] Two polymers were chosen to prepare high sulfur content polymeric nanoparticles; the product of the copolymerization of sulfur and perillyl alcohol (S-PA) and the product of the copolymerization of sulfur and geraniol (S-Ger). Perillyl alcohol and geraniol are both naturally derived terpenes that are found in the essential oils of lavender and geranium, among others. Copolymerization of elemental sulfur with perillyl alcohol or geraniol was carried out by adding the terpene monomers to molten sulfur at 135 °C and further heating at 175 °C until the mixture became homogeneous and viscous. The reaction mixture was cured overnight at 140 °C. Following curing, the polymers were ground into powders using a pestle and mortar. ¹ H NMR and FTIR spectroscopy were used to evaluate the reaction by probing the signals corresponding to alkene units of the monomer. The Tg of the resultant materials was determined by DSC. DSC was also used to determine the presence of any unreacted crystalline sulfur within the materials. The reaction of sulfur with geraniol yielded black pliable materials that become brittle after refrigeration (97% yield). DSC analysis of S50-Ger showed a Tg of 5.8 °C, indicating the presence of a polymeric material.

Formulation of High Sulfur Content Polymer Nanoparticles

[00180] The solubility of S-PA and S-Ger in chloroform and THF is ideal for the formulation of nanoparticles by techniques that require preformed polymers. The solubility of both polymers in these solvents also allowed for two methods for nanoparticle formulation to be investigated, namely, the nanoprecipitation method and the emulsion-solvent evaporation method. S50-Gerwas used to formulate nanoparticles by both methods. Water was chosen as the antisolvent for both methods. The dispersions were analysed by dynamic light scattering (DLS), where the main parameters measured were the z-average diameter and the polydispersity index (PDI).

[00181] An emulsion-solvent evaporation method for the preparation of polymeric nanoparticles was investigated for S-Ger. For this method, an oil-in-water (chloroform/water) emulsion was formed whereby the polymer was dissolved in the oil phase and a suitable surfactant was dissolved in the water phase. Various emulsifiers were trialled, including Tween 80, poly(vinyl alcohol) (PVA), Brij S20, and sodium dodecyl sulfate (SDS). Tween 80 and Brij S20 are both non-ionic surfactants with a hydrophile-lipophile balance of 15; PVA is a non-ionic polymer that is often used as an emulsifier whereas SDS in an anionic surfactant.

[00182] The trial formulation for the preparation of S50-Ger dispersions consisted of a concentration of 5 mg/mL of polymer in chloroform and 10 mg/mL of surfactant in water, with an oil to water ratio of 1 :9. Stable emulsions were formed with all surfactants trialled, which were then allowed to stir at room temperature to allow for the evaporation of chloroform. The resulting dispersions were cream coloured with no visible aggregates. DLS analysis of the dispersions formed with both Brij S20 and SDS were multimodal with poor quality correlograms suggesting particle aggregation; thus, Brij S20 and SDS were deemed unsuitable as surfactants for the preparation of S50-Ger nanoparticles. DLS of the nano dispersions of S50-Ger prepared with PVA as an emulsifier gave monomodal distributions at various dilutions with a z-average diameter of 367.7 nm and a PDI of 0.173. The dispersions were found to be stable for up to 14 days.

[00183] A nanoprecipitation method was also investigated for inverse vulcanized polymer nanoparticles; THF was chosen as the solvent for the polymer and water as the antisolvent. The nanoprecipitation method does not require the use of a surfactant; therefore, a surfactant-free dispersion was formulated to establish whether S50-Ger polymer nanoparticles could be formed. The trial formulation consisted of 5 mg/mL polymer dissolved in THF, with a THF to water ratio of 1 :9. After evaporation of THF, a cream coloured cloudy solution remained with no visible aggregates. The size distribution by intensity obtained by DLS indicates the successful synthesis of polymeric nanoparticles with monomodal distributions. The particles obtained have a z-average diameter of 138 nm and a relatively low PDI of 0.165. The effect of the concentration of polymer dissolved in THF was studied by employing concentrations of 5, 10, and 20 mg/mL. The smallest particles were obtained with the lowest concentrations of polymer, and increasing the concentration results in an increase in both the nanoparticle z-average diameter and the PDI.

Antibacterial Activity

[00184] The antibacterial activity of S-polymer nanoparticles was assessed against Grampositive methicillin-resistant S. aureus (strain LISA300) and Gram-negative P. aeruginosa (strain PAO1). Nanoparticles were incubated in media containing bacteria. Viable cells were enumerated to assess whether the nanoparticles induce an antibacterial effect against the cells compared to a blank solution that was prepared by dropping THF into water and allowing the solution to stir overnight at room temperature. The preparation of the blank followed the same procedure as that used in the preparation of the polymer nanoparticles, however, with no polymer dissolved in the solvent phase. Polymer nanoparticles were tested at various concentrations in order to investigate the dose-response relationship. S50-PA nanoparticles at concentrations of 14, 27, 55, 220, and 440 pg/mL were tested over a period of 5 h (Figure 17a) against S. aureus in nutrient-rich LB medium, within which bacteria grow exponentially. After 5 h, a 1.07 (>90%) and 3.2 log (>99.9%) reduction in the number of viable cells compared to the blank was achieved for 14 and 440 pg/mL of nanoparticles, respectively. The higher concentrations of 220 and 440 pg/mL were further tested after 24 h and were found to have reduced the number of viable cells by 1 .09 log (>90%), compared to the blank. The minimum inhibitory concentration (MIC) of S50-PA nanoparticles was evaluated during a 24 h incubation period with methicillin-resistant S. aureus (Figure 18). A 50% growth inhibition (MICso) in S. aureus, compared to untreated samples, was found at a nanoparticle concentration of 64 pg/mL (p < 0.0001 relative to untreated control), and 90% inhibition (MICgo) was achieved at 512 pg/mL (p < 0.0001 relative to untreated control).

[00185] S50-Ger nanoparticles formulated in the same way were also found to have an inhibitory effect against S. aureus after a 5 h incubation period (Figure 19). When the initial S. aureus concentration was lowered from approximately 100,000 CFU/mL (5 log CFU/mL) to 100 CFU/mL (2 log CFU/mL) (Figure 20), a reduction in the number of viable cells following addition of nanoparticles was apparent, compared to the initial culture, indicating that the S50-PA nanoparticles exert a bactericidal effect. That is, the particles kill bacterial cells, rather than merely suppressing cell growth. The effect of additional dosing of nanoparticles was also investigated, whereby the cultures were spiked with an additional 100 pL dose of particles or control after 2 h of incubation, the effect was enumerated after 5 h and it was found that the additional dose reduced the number of viable cells further.

[00186] S50-PA polymeric nanoparticles were tested against P. aeruginosa strain PAO1 in nutrient-rich LB broth for a period of 5 h (Figure 17b) at final concentrations of 220 and 440 pg/mL. After 5 h incubation both concentrations of nanoparticles achieved a 2.9 log (>99 %) reduction in viable P. aeruginosa cells compared to the control sample. In a 24 hour assay, the MICso of S50-PA nanoparticles for P. aeruginosa PAO1 was 128 pg/mL (p < 0.0001 relative to untreated control) (Figure 21). This shows that S50-PA polymeric nanoparticles exhibit an antibacterial effect against both S. aureus and P. aeruginosa Similarly, S50-Ger nanoparticles were tested against P. aeruginosa PAO1 (Figure 22) and were also found to show an inhibitory effect, demonstrating the antibacterial properties of the polymer nanoparticles.

[00187] The potential of using high sulfur content nanoparticles as combination therapies with antibiotics was investigated. Nanoparticles were tested against an extensively drugresistant P. aeruginosa strain (B9) in combination with tobramycin, an aminoglycoside antibiotic that is used to treat complicated infections such as septicaemia and urinary tract infections and to manage P. aeruginosa infections in people with cystic fibrosis. The effect of varying the tobramycin concentration (1-512 pg/mL) whilst maintaining a nanoparticle concentration of 128 pg/ml was investigated (Figure 23). In the absence of antibiotics, 512 ug/mL of nanoparticles were required to inhibit >50% of the growth (p < 0.0001 relative to untreated control) of the highly drug-resistant P. aeruginosa B9 strain. With treatment of tobramycin alone, B9 growth was completely inhibited only at the top concentration of 512 pg/mL (p < 0.0001 relative to untreated control), however, with the addition of nanoparticles the growth was completely inhibited at a concentration of 256 pg/mL (p < 0.0001 relative to untreated control). Furthermore, reductions in growth with dual nanoparticle plus tobramycin treatment, relative to treatment with tobramycin alone, were observed at all tobramycin concentrations between 16 and 128 pg/mL.

Anti biofilm Activity

[00188] Biofilms provide favourable conditions for bacteria as they offer protection from the immune system of the host, exhibit phenotypic resistance to antimicrobial agents, and ensure efficient distribution of resources throughout the microbial population. These factors make biofilm bacteria more challenging than planktonic cells. The ability of S50-PA nanoparticles to inhibit biofilm formation on the surface of a container was evaluated against S. aureus LISA300 and P. aeruginosa PAO1. Nanoparticles were added to vials containing bacterial culture and were statically incubated for 24 and 48 h, a control sample was prepared by adding water to the culture instead of nanoparticles. S50-PA nanoparticles were found to inhibit S. aureus biofilm formation over 48 h (Figure 17c).

Cytotoxicity

[00189] The cytotoxicity of high sulfur content polymeric nanoparticles against the liver carcinoma cell line HepG2 was investigated using the MTT assay.

[00190] In brief, the nanoparticles were added to approximately 80% confluent HepG2 cell cultures and were incubated for 24 h. Untreated cells were used as the negative control. After incubation, the culture media was removed and replaced with fresh medium containing MTT labelling agent. The solutions were incubated for 4 h, before the solubilizing agent was added. The absorbance at 595 nm was measured after leaving the solutions in the presence of the solubilizing agent to incubate overnight. S50-PA nanoparticles at final concentrations of 14, 27, 55, 220 and 440 pg/mL were evaluated for their cytotoxicity against HepG2 cells. The cytotoxicity of the nanoparticles was expressed as % cell viability compared to untreated HepG2 cells (Figure 17d). More than 80% cell viability was observed at all nanoparticle concentrations tested and no clear dose-dependent toxicity was observed.

[00191] While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims. REFERENCES

1. M. Schmidt, in New Uses of Sulfur, 1978, pp. 1-12.

2. M. Sedivy, J. Mod. Ital. Stud., 2011 , 16, 1-18.

3. D. W. Thomson, 1840, 163-180.

4. J. J. Griebel, R. S. Glass, K. Char and J. Pyun, Prog. Polym. Sci., 2016, 58, 90-125.

5. M. J. H. Worthington, R. L. Kucera and J. M. Chalker, Green Chem., 2017, 19, 2748- 2761.

6. W. J. Chung, J. J. Griebel, E. T. Kim, H. Yoon, A. G. Simmonds, H. J. Ji, P. T. Dirlam, R. S. Glass, J. J. Wie, N. A. Nguyen, B. W. Guralnick, J. Park, A. Somogyi, P. Theato, M. E. Mackay, Y. Sung, K. Char and J. Pyun, Nat. Chem., 2013, 5, 518-524.

7. M. P. Crockett, A. M. Evans, M. J. H. Worthington, I. S. Albuquerque, A. D. Slattery, C. T. Gibson, J. A. Campbell, D. A. Lewis, G. J. L. Bernardes and J. M. Chalker, Angew. Chemie Int. Ed., 2016, 55, 1714-1718.

8. D. J. Parker, H. A. Jones, S. Petcher, L. Cervini, J. M. Griffin, R. Akhtar and T. Hasell, J. Mater. Chem. A, 2017, 5, 11682-11692.

9. B. Zhang, L. J. Dodd, P. Yan and T. Hasell, React. Fund. Polym., 2021 , 161 , 104865.

10. I. Gomez, O. Leonet, J. A. Blazquez and D. Mecerreyes, ChemSusChem, 2016, 9, 3419-3425.

11. C. Herrera, K. J. Ysinga and C. L. Jenkins, ACS Appl. Mater. Interfaces, 2019, 11, 35312-35318.

12. M. Arslan, B. Kiskan, E. C. Cengiz, R. Demir-Cakan and Y. Yagci, Eur. Polym. J., 2016, 80, 70-77.

13. D. J. Parker, S. T. Chong and T. Hasell, RSC Adv., 2018, 8, 27892-27899.

14. J. A. Smith, X. Wu, N. G. Berry and T. Hasell, J. Polym. Sci. Part A Polym. Chem., 2018, 56, 1777-1781.

15. B. Zhang, H. Gao, P. Yan, S. Petcher and T. Hasell, Mater. Chem. Front., 2020, 4, 669- 675.

16. X. Wu, J. A. Smith, S. Petcher, B. Zhang, D. J. Parker, J. M. Griffin and T. Hasell, Nat. Commun., 2019, 10, 647.

17. D. A. Boyd, V. Q. Nguyen, C. C. McClain, F. H. Kung, C. C. Baker, J. D. Myers, M. P. Hunt, W. Kim and J. S. Sanghera, ACS Macro Lett., 2019, 8, 113-116. 18. J. Kuwabara, K. Oi, M. M. Watanabe, T. Fukuda and T. Kanbara, ACS Appl. Polym. Mater., 2020, 2, 5173-5178.

19. O. Bayram, B. Kiskan, E. Demir, R. Demir-Cakan and Y. Yagci, ACS Sustain. Chem. Eng., 2020, 8, 9145-9155.

20. J. A. Smith, R. Mulhall, S. Goodman, G. Fleming, H. Allison, R. Raval and T. Hasell, ACS Omega, 2020, 5, 5229-5234.

21. R. A. Dop, D. R. Neill and T. Hasell, Biomacromolecules, 2021 , 22, 5223-5233.

22. M. Mann, J. E. Kruger, F. Andari, J. McErlean, J. R. Gascooke, J. A. Smith, M. J. H. Worthington, C. C. C. McKinley, J. A. Campbell, D. A. Lewis, T. Hasell, M. V. Perkins and J. M. Chalker, Org. Biomol. Chem., 2019, 17, 1929-1936.

23. S. Petcher, B. Zhang and T. Hasell, Chem. Commun., 2021 , 57, 5059-5062.

24. J. M. Scheiger, C. Direksilp, P. Falkenstein, A. Welle, M. Koenig, S. Heissler, J. Matysik, P. A. Levkin and P. Theato, Angew. Chemie - 1 nt. Ed., 2020, 59, 18639-18645.

25. L. A. Limjuco, H. T. Fissaha, H. Kim, G. M. Nisola and W. J. Chung, ACS Appl. Polym. Mater., 2020, 2, 4677-4689.

26. A. G. Simmonds, J. J. Griebel, J. Park, K. R. Kim, W. J. Chung, V. P. Oleshko, J. Kim, E. T. Kim, R. S. Glass, C. L. Soles, Y. E. Sung, K. Char and J. Pyun, ACS Macro Lett., 2014, 3, 229-232.

27. M. Mann, B. Zhang, S. J. Tonkin, C. T. Gibson, Z. Jia, T. Hasell and J. M. Chalker, Polym. Chem., , DGI:10.1039/D1 PY01416A.

28. M. Thielke, L. Bultema, D. Brauer, B. Richter, M. Fischer and P. Theato, Polymers (Basel)., 2016, 8, 266.

29. S. Petcher, D. J. Parker and T. Hasell, Environ. Sci. Water Res. Technol., 2019, 5, 2142- 2149.

30. T. Hasell, D. J. Parker, H. A. Jones, T. McAllister and S. M. Howdle, Chem. Commun., 2016, 52, 5383-5386.

31. I. J. Joye and D. J. McClements, Trends Food Sci. Technol., 2013, 34, 109-123.

32. M. E. Matteucci, M. A. Hotze, K. P. Johnston and R. O. Williams, Langmuir, 2006, 22, 8951-8959.

33. C. Hogarth, K. Arnold, A. McLauchlin, S. P. Rannard, M. Siccardi and T. O. McDonald, J. Mater. Chem. B, 2021 , 9, 9874-9884. 34. J. M. Taylor, K. Scale, S. Arrowsmith, A. Sharp, S. Flynn, S. Rannard and T. O. McDonald, Nanoscale Adv. , 2020, 2, 5572-5577.

35. T. O. McDonald, L. M. Tatham, F. Y. Southworth, M. Giardiello, P. Martin, N. J. Liptrott, A. Owen and S. P. Rannard, J. Mater. Chem. B, 2013, 1 , 4455-4465. 36. J. A. Smith, S. J. Green, S. Petcher, D. J. Parker, B. Zhang, M. J. H. Worthington, X.

Wu, C. A. Kelly, T. Baker, C. T. Gibson, J. A. Campbell, D. A. Lewis, M. J. Jenkins, H. Willcock, J. M. Chalker and T. Hasell, Chem. - A Eur. J., 2019, 10433-10440.

37. M. Mann, X. Luo, A. Tikoalu, C. T. Gibson, Y. Yin, R. Al-attabi, G. G. Andersson, C. L. Raston, L. C. Henderson, A. Pring, T. Hasell and J. M. Chalker, Chem. Common., , D0l:10.1039/d1cc01555a.

38. A. D. Tikoalu, N. A. Lundquist and J. M. Chalker, Adv. Sustain. Syst., 2020, 4, 1-9.

39. C. Liu, J. Peng, L. Zhang, S. Wang, S. Ju and C. Liu, J. Clean. Prod., 2018, 196, 109- 121.

40. J. S. M. Lee, D. J. Parker, A. I. Cooper and T. Hasell, J. Mater. Chem. A, 2017, 5, 18603- 18609.

41. W. H. O. WHO, WHO, 2005, WHO/SDE/WS, WHO/SDE/WSH/05.08/10.