Sulfonated particles

Abstract

The present disclosure relates to particles formed from one or more block co-polymers having the structure: L-[A]x-b-[B]y-L, wherein A is a monomer residue comprising at least one sulfonate or sulfate substituent, B is a hydrophobic monomer residue, L is a capping group, x is greater than 3, and y is 25 to 200. The disclosure further relates to compositions and pharmaceutical compositions comprising said particles, devices comprising said compositions, methods of using said compositions, and said pharmaceutical compositions for use in the prevention or treatment of viral infections.

Description

[0001] Sulfonated Particles

[0002] Field of the Invention

[0003] The present invention relates to particles, compositions and pharmaceutical compositions comprising said particles, devices comprising said compositions, methods of using the composition and said pharmaceutical compositions for use in the prevention or treatment of viral infections.

[0004] Background of the Invention

[0005] Viruses have profound impacts on human and animal health, on account of their potential for significant morbidity and mortality[l]. Various approaches exist to address the global threat posed by viruses. Prevention, typically using vaccination, is the most effective method to halting viral spread. [2] However, in certain instances, particularly with newly discovered viruses or viral variants, vaccines do not exist or viruses can mutate around the protection vaccines provide. This necessitates the development of antiviral agents, including the exploration of new classes of antivirals. Whilst typical antivirals take the form of small molecule drugs, that act via an intracellular mechanism [3, 4], there are well known macromolecules with potent extracellular antiviral properties [5-7],

[0006] Macromolecular antivirals typically act directly on the virus, without the need for internalisation by the cell. [8] They often demonstrate broad-spectrum activity, which is highly advantageous. [9] These macromolecular antivirals usually act as mimics of cellsurface receptors, such as heparan sulfate proteoglycans (HSPGs) or sialic acids, which are highly conserved across different mammalian cell types.

[0010] These cell-surface receptors have been widely studied and are utilized by a wide range of viruses as initial attachment receptors in their infection cycle. [11-13] Macromolecules that mimic HSPGs act as competitors to these receptors on the cell surface, preventing virus attachment and subsequent viral entry

[0014] . The use of highly sulfonated materials acting as HSPG mimics have been widely studied, using a range of materials, including polymers [9, 15-17], nanoparticles [18, 19], nanogels

[0020] , dendrimers

[0021] , macrocycles [22, 23] and polymer-coated nanoparticles. [24, 25] However, only a few of these antivirals have made their way to clinical trials and in some instances have been shown to increase infection rates.

[0026] Typically these types of sulfonated macromolecules have a reversible binding interaction with viruses, termed virustatic [27-29] , This reversible, non-destructive, interaction with viruses limits their clinical use. Conversely, virucidal materials confer a conformational change in the virion post interaction, resulting in an irreversible destruction of the virus, often releasing the viral genomic material). [28, 30, 31] This renders the virus inert and unable to infect. Most commercially available virucides are associated with being highly cytotoxic, such as bleach and alcohols, [32-34] stopping their therapeutic use. Harnessing a virucidal mode of action, in a biocompatible material, could therefore be of significant societal benefit.

[0007] Nanoparticles containing a ‘core’ and a ’corona’, capable of multi-valent interactions, have recently been shown to be capable of accessing a virucidal mode of action, following coating of an already formed core. [22, 24, 25, 35-38]

[0008] Being able to produce the core / corona architecture in situ has advantages over such approaches, including greater control over both core and corona size and composition. Sterically-stabilised polymer nanoparticles with a distinct core and solvated corona are inherently multi -valent and can readily be produced using a wide range of approaches. A powerful technique for preparing sterically-stabilised block copolymer nanoparticles with tuneable core diameters, shell thicknesses, morphologies, and chemical functionality is polymerization-induced self-assembly (PISA). PISA utilises a macromolecular chain-transfer agent (Macro-CTA), which is typically hydrophilic which is chain-extended via the growth of a hydrophobic polymer chain

[0039] . As the hydrophobic chains become longer, they self-assemble forming polymer nanoparticles, where the hydrophilic polymer forms a corona around the hydrophobic core. The size of both the core and polymer corona can be readily controlled during PISA, through varying parameters such as the degree of polymerization of the core and corona blocks, the concentration of the polymerization and the solvent quality of the polymerization medium [40, 41] , Summary of the Invention

[0009] The present inventors have identified a sulfonated particle with antiviral properties.

[0010] In a first aspect the present invention is directed to a particle formed from one or more block co-polymers having the structure:

[0011] L-[A]x-Z>-[B]y-L

[0012] Wherein:

[0013] A is a monomer residue comprising at least one sulfonate substituent or sulfate substituent,

[0014] B is a hydrophobic monomer residue, L is a capping group, x is greater than 5, and y is 25 to 200.

[0015] In a second aspect the invention is directed to a composition comprising a plurality of particles of the first aspect.

[0016] In a third aspect the invention is directed to method of sterilisation or viral disinfection, comprising using an effective amount of the composition of the second aspect.

[0017] In a fourth aspect the invention is directed to a device for sterilisation or viral disinfection comprising the composition of the second aspect and a means for dispensing the composition.

[0018] In a fifth aspect the invention is directed to a pharmaceutical composition comprising a plurality of particles of the first aspect and one or more pharmaceutically acceptable excipients.

[0019] In a sixth aspect, the invention is directed to a pharmaceutical composition of the fifth aspect for use in the prevention or treatment of viral infections. Detailed Description of the Invention

[0020] Definitions

[0021] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0022] A “therapeutically effective amount” means the amount of the particle of the invention that, when administered to a mammal for treating a disease, is sufficient to affect such treatment for the disease. The "therapeutically effective amount" will vary depending on the particle, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0023] The term “HSPG-binding virus” refers to a virus which is known to be, or is believed to be, capable of binding to heparan sulfate proteoglycans (HSPGs) on the surface of host cells after the virus has infected a host subject.

[0024] The terms “sulfonate” and “sulfate” refer to the following anionic substituent groups and encompass all salts and associated counterions thereof:

[0025] As the skilled person will be aware, in block co-polymer notation “6” indicates that the polymer is a diblock co-polymer. N-6-M therefore indicates that the polymer is a diblock co-polymer of polymer N and polymer M.

[0026] Particles of the Invention

[0027] The present invention is directed to a particle formed from one or more block copolymers having the structure of Formula I:

[0028] L-[A]x-Z>-[B]y-L

[0029] Formula I

[0030] Wherein:

[0031] A is a monomer residue comprising at least one sulfonate or sulfate substituent, B is a hydrophobic monomer residue,

[0032] L is a capping group, x is greater than 3, and y is 25 to 200.

[0033] A may have at least one sulfonate substituent. A may have the structure of Formula II:

[0034] Formula II Wherein R1is aryl, Ci-2oalkyl, Ci-2oalkylene-aryl, C(O)OR4, C(O)NHR4, OC(O)R4, NHC(O)R4or sulfonate, R2and R3are independently selected from hydrogen and Ci-4 alkyl; and R4is C i-io alkyl or aryl. Each of the aryl, Ci-2oalkyl, Ci-2oalkylene—aryl, or R. groups is substituted with one or more sulfonate or sulfate groups, and is optionally substituted with one or more substituents selected from hydroxy, halo, Ci-4 alkyl, Ci-4 alkoxy, aryl and cyano.

[0035] Where A has the structure of Formula II, R1may be selected from: Wherein K is Ci-nalkylene.

[0036] In a most preferred embodiment, A may be a monomer residue derived from polymerisation of styrene sulfonate (SS).

[0037] B is a hydrophobic monomer. Polymer block [B]yforms the core of the particle. Therefore, by hydrophobic monomer it is meant that the block co-polymer forms a colloid in water wherein Polymer [B]yforms the core of the colloid. Hydrophobic monomers are well known in the art.

[0038] For example, B may be selected from a monomer residue having at least one substituent selected from aryl, branched or linear alkyl, branched or linear allyl, fluorinated alkyl, fluorinated aryl.

[0039] B may be selected from a monomer residue derived from polymerisation of styrenic compounds, methacrylic compounds, acrylamide compounds, fluorinated compounds, or compounds having a Ci-Cioalkyl group or combinations thereof.

[0040] B may be selected from a monomer residue derived from polymerisation of benzyl methacrylate (BzMA), methyl methacrylate, glycerol methacrylate, ethyl methacrylate, 2-ethylhexyl methacrylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate, 2,2,2-trifluoroethyl methacrylate, lauryl methacrylate, allyl methacrylate, octyl methacrylate, t-butyl methacrylate, tert-butyl acrylate, diacetone acrylamide, lactic acid, dimethyl siloxane, styrene, or combinations thereof.

[0041] In a preferred embodiment, B may be a monomer residue derived from polymerisation of benzyl methacrylate, styrene, glycerol methacrylate, diacetone acrylamide, methyl methacrylate, tert-butyl acrylate or combinations thereof. In a most preferred embodiment, B may be a monomer residue derived from polymerisation of benzyl methacrylate.

[0042] Therefore, in some embodiments of the particle formed from one or more block copolymer having the structure of Formula I: L-[A]x-6-[B]y-L

[0043] Formula I

[0044] A is a monomer residue derived from polymerisation of styrene sulfonate and B is a monomer residue derived from polymerisation of benzyl methacrylate (BzMA), styrene, glycerol methacrylate, diacetone acrylamide, methyl methacrylate, tert-butyl acrylate or combinations thereof.

[0045] In some embodiments the particle is formed from one or more block co-polymer having the structure of Formula III.

[0046] L-(poly(styrene sulfonate))x-6-(poly(benzyhnethacrylate))y-L Formula III

[0047] Polymers of styrene sulfonate (SS) may be referred to as PSS. Polymers of benzyl methacrylate (BzMA) may be referred to as PBzMA. Formula (III) is therefore also referred to as PSSx-PBzMAy. x defines the degree of polymerisation of the [A] block, x is greater than 3. x may be greater than 5. x may be greater than 12. In some embodiments, x is from 5 to 100, such as from 12 to 70 or from 40 to 60. y defines the degree of polymerisation of the [B] block, y is from 25 to 200. y may be from 25 to 175, from 25 to 250, from 25 to 125, from 25 to 100 or from 25 to 75. y may be from 30 to 200, from 35 to 200 or from 40 to 200. In some preferred embodiments, y is from 25 to 75, such as from 30 to 70 or from 35 to 65. Herein, the particle may be referred to herein by the degree of polymerization (DP) of each block. For example, PSS47-PBZMA50 will be referred to as 47:50.

[0048] In some embodiments the particle is formed from one or more block co-polymer having the structure of Formula IV:

[0049] L-(poly(styrene sulfonate))i2-70-6-(poly(benzylmethacrylate))30-70-L

[0050] Formula IV In some embodiments the particle is formed from one or more block co-polymer having the structure of Formula V:

[0051] L-(poly(styrene sulfonate))i2-6-(poly(benzylmethacrylate))50-L

[0052] Formula V

[0053] In some embodiments the particle is formed from one or more block co-polymer having the structure of Formula VI:

[0054] L-(poly(styrene sulfonate))47-6-(poly(benzylmethacrylate))50-L

[0055] Formula VI

[0056] In some embodiments the particle is formed from one or more block co-polymer having the structure of Formula VII:

[0057] L-(poly(styrene sulfonate))47-6-(poly(benzylmethacrylate))ioo-L

[0058] Formula VII

[0059] The particle may be a nanoparticle. The particle may have a number-average particle diameter of 100 nm to 400 nm, such as from 150 to 400 nm, from 170 nm to 270 nm or from 190 nm to 240 nm.

[0060] Number-average particle diameters may be determined using transmission electron microscope (TEM), such as using transmission electron microscope (TEM) images by analysing 200 particles using ImageJ software.

[0061] The particle may have a mean hydrodynamic diameter of from 150 to 950 nm, such as from 150 nm to 800 nm, from 150 nm to 600 nm, from 150 nm to 500 nm, from 150 nm to 400 nm, from 150 nm to 300 nm, from 170 nm to 270 nm or from 190 nm to 240 nm. Mean hydrodynamic diameters are obtained via DLS for nanoparticles after a freezethaw cycle and then redispersion in water.

[0062] The number of copolymer chains present in a nanoparticle can be represented by the mean aggregation number (Nagg) and calculated using Equation 1, [44-46]

[0063] Equation 1 where xi0 / is the fraction of solvent in the core of the nanoparticle, Rcoreis the radius of the nanoparticle core, and N chain is the volume occupied by a single copolymer chain in the core of nanoparticle. Rcoremay be determined from D„, xi0 / may be assumed to be zero, and N chain may be calculated using the density of the [B] polymer, and the target copolymer composition. The particle of the present invention may have a Naggof from 0.5 x 105to 5 x 105, such as from 0.75 x 105to 4.5 x 105or 1 x 105to 4 x 105or 1.25 x 105to 3.5 x 105. In preferred embodiments the particle has a Naggof from 1 x 105to 3.5 x 105, most preferably from 1.25 x 105to 2 x 105.

[0064] Naggmay be extended to determine the average number of copolymer chains per unit surface area of a nanoparticle (Sogg), using Equation 2.

[0065] Equation 2

[0066] The particle of the present invention may have a Saggof from 0.005 to 0.05, for example 0.01 to 0.04. In preferred embodiments the particle has a Saggof from 0.1 to 0.3, most preferably from 0.1 to 0.2.

[0067] L is a capping group and may be any suitable capping group. In some embodiments, each L group may be independently selected from hydrogen, hydroxyl, bromo, chloro, alkyl or one of the following groups:

[0068]

[0069] Wherein:

[0070] R5is hydrogen, halo, cyano, CO2H, Ci-shaloalkyl, Ci-salkylene-OH, or Ci-3alkylene- NH2; R6and R7are independently selected from hydrogen, and Ci-3alkyl optionally substituted with cyano, halo or CO2H;

[0071] R8is S-Ci -isalkyl, S-aryl, NR9R10 or aryl, said Ci-isalkyl any aryl groups being optionally substituted with one or more substituents selected from hydroxy, Ci-3alkyl, halo, cyano, CO2H, and Ci-3haloalkyl; R9is hydrogen or Ci-3alkyl; and

[0072] R10is hydrogen, C1-12 or aryl, said C1-12 alkyl and aryl groups being optionally substituted with one or more substituents selected from hydroxy, Ci-3alkyl, halo, cyano, CO2H, and Ci-3haloalkyl. In some embodiments, each L group may be independently selected from hydrogen, hydroxyl, bromo, chloro, alkyl or one of the following groups: In some embodiments, each L group may be independently selected from one of the following groups:

[0073] The particles of the present invention can be formed by any suitable standard polymerisation process as may be determined by the skilled person. Such processes include living polymerisation techniques. For example, block co-polymer particles of the present invention may be prepared by reversible addition fragmentation chaintransfer (RAFT) PISA in ethanol / water mixtures following a previously reported related protocol for poly(potassium 3-sulfopropyl methacrylate)functional PBzMA particles.

[0043] Other suitable methods of preparing the particles of the present invention include ring-opening metathesis polymerization-induced self-assembly (ROMPISA).

[0074] Compositions

[0075] The particles presented herein have virucidal properties which make them useful for a number of applications.

[0076] The present invention therefore further relates to compositions comprising the particle of the present invention. Pharmaceutical compositions comprising the particle of the present invention have applications in the prevention and treatment of viral infections in humans and other animals. Such pharmaceutical compositions are described separately below.

[0077] Other applications include the sterilisation and disinfection of surfaces contaminated, or suspected to be contaminated, by viruses.

[0078] There is therefore provided a composition comprising a plurality of particles of the present invention. The particles of the present invention are present in an effective amount, which is an amount sufficient for destroying or immobilizing viruses. The compositions comprise a carrier, which may be a stabiliser, wetting agent, emulsifier, thickener, fragrance, colourant or mixture thereof.

[0079] The composition may be formulated as a solution, a gel, a foam, or an emulsion. In one embodiment the composition is a sterilising solution or a disinfecting solution.

[0080] Pharmaceutical Compositions

[0081] The particles of the present invention may be formulated into pharmaceutical compositions. Said pharmaceutical compositions may be administered to a patient.

[0082] There is therefore provided a pharmaceutical composition comprising a plurality of particles of the present invention. The pharmaceutical composition may be in a form suitable for oral use, for example as tablets, capsules, caplets, pills, troches, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and cachets. The pharmaceutical composition may be in a form suitable for topical use, for example as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels. The pharmaceutical composition may be in a form suitable for transdermal administration such as via transdermal patches. The pharmaceutical composition may be in a form suitable for administration by inhalation, for examples as a dry powder, aerosol, suspension, or solution. The pharmaceutical composition may be in a form suitable for administration by insufflation, or for parenteral administration.

[0083] As used herein, "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled such that interactions which would substantially reduce the efficacy of the particle of the invention when administered to a patient and interactions which would result in pharmaceutical compositions that are not pharmaceutically acceptable are avoided. In addition, each excipient must of course be of sufficiently high purity to render it pharmaceutically acceptable.

[0084] Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carrying or transporting of the particle or particles of the invention once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.

[0085] Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, hemectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents.

[0086] The pharmaceutical compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company).

[0087] The pharmaceutical composition may be in a solid oral dosage form such as a tablet or capsule comprising a safe and effective amount of a particle of the invention and a diluent or filler. Suitable diluents and fillers include lactose, sucrose, dextrose, mannitol, sorbitol, starch (e.g. corn starch, potato starch, and pre-gelatinized starch), cellulose and its derivatives (e.g. microcrystalline cellulose), calcium sulfate, and dibasic calcium phosphate. The oral solid dosage form may further comprise a binder. Suitable binders include starch (e.g. corn starch, potato starch, and pre-gelatinized starch), gelatin, acacia, sodium alginate, alginic acid, tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g. microcrystalline cellulose). The oral solid dosage form may further comprise a disintegrant. Suitable disintegrants include crospovidone, sodium starch glycolate, croscarmelose, alginic acid, and sodium carboxymethyl cellulose. The oral solid dosage form may further comprise a lubricant. Suitable lubricants include stearic acid, magnesium stearate, calcium stearate, and talc.

[0088] The pharmaceutical composition may be in a dosage form adapted for administration to a patient parenterally including subcutaneous, intramuscular, intravenous, intrathecal or intradermal. Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the composition isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents. The composition may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0089] The pharmaceutical composition may be in a dosage form adapted for topical administration to a patient. For example, the particle of the invention may be applied topically as a cream, an ointment, a lotion, a solution, a paste, a spray, a foam, or a gel. Suitable topical compositions may typically comprise one or more of emollients, solubilising agents, humectants, gelling agents, preservatives, permeation enhancers, chelating agents, antioxidants, buffering agents and solvents.

[0090] The pharmaceutical composition may be in a dosage form adapted for administration to a patient by inhalation (e.g. nasal or pulmonary). For example, the particle of the invention may be inhaled into the lungs as a dry powder, an aerosol, a suspension, or a solution. For administration by inhalation, the pharmaceutical compositions can be conveniently delivered in the form of an aerosol spray, from pressurized packs or via a nebulizer, or with the use of suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, propane, butane or other suitable gases or mixture of gases. The particle size of the aerosol is not critical, but particles under about 3-5 microns in diameter are convenient for deep penetration into the pulmonary system. Continuous administration in a hospital environment or periodic administration, e.g. at 1 to 6 hour intervals, will typically allow the antiviral particles as described herein to act effectively in the subject's pulmonary system.

[0091] An effective amount of a particle of the present invention for use in therapy of proliferative disease is an amount sufficient to symptomatically relieve in a warmblooded animal, particularly a human, the symptoms of infection, to slow the progression of infection, or to reduce in patients with symptoms of infection the risk of getting worse.

[0092] The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the route of administration. For example, the pharmaceutical composition intended for oral administration to humans will generally contain, for example, from 0.5 mg to 0.5 g of active agent, which is the particle of the present invention.

[0093] The size of the dose for therapeutic or prophylactic purposes of a particle of the present invention will naturally vary according to the viral infection being treated, the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

[0094] Therapeutic Uses and Applications

[0095] The particles of the invention have been found to have antiviral activity. As a consequence, they are potentially useful in the treatment of viral infections.

[0096] The particles of the present invention are either virustatic or virucidal. As previously discussed, virustatic materials make contact with the virus and prevent viral infection of cells via a reversible process. Changes in pH, temperature or dilution can result in the disassociation of the antiviral from the virus, leaving an intact, infectious virion. Conversely, virucidal materials make direct contact with the viral particle and irreversibly destroys the virus upon contact, rendering it inert and unable to infect. There is therefore provided a pharmaceutical composition comprising a particle of the present invention or a pharmaceutically acceptable salt or salts thereof, or a pharmaceutical composition as defined herein for use in the prevention or treatment of viral infections.

[0097] Heparan sulfate proteoglycans (HSPGs) are proteoglycan receptors located on the surface of almost all eukaryotic cells. Many extracellular proteins bind to cell-surface HSPGs. A number of viruses also utilise HSPGs as attachment factors. These viruses typically have capsid proteins capable of binding to HSPGs on the surface of host cells, which leads to viral entry of the host cell. It is postulated that the particles of the present invention are able to mimic the HSPG-viral interaction and that they preferentially bind to and destroy HSPG-binding viruses. Therefore, in another aspect, the present invention provides a particle of the present invention, or a pharmaceutically acceptable salt or salts thereof, or a pharmaceutical composition as defined herein, for use in the prevention or treatment of viral infections associated with one or more HSPG-binding viruses.

[0098] HSPG-binding viruses include, but are not limited to, herpes simplex virus (HSV), adenovirus, adeno-associated virus, human papillomavirus (HPV), respiratory syncytial virus (RSV), dengue virus, norovirus, lentivirus, human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), human metapneumovirus (HMPV), human parainfluenza virus type 3 (HPIV-3), coronavirus (such as MERS-CoV, SARS-CoV, or SARS-CoV-2), foot-and-mouth disease virus, hepatitis B virus, hepatitis C virus, Ebola virus, nipah virus, Rift Valley fever virus, West Nile virus, Crimean Congo virus, Toscana virus, ZIKA virus, Chickungunya virus (CHIKV), Akabane virus (AKAV), and Schmallenberg virus (SBV).

[0099] Coronaviruses include alpha, beta, gamma, and delta coronaviruses. The present invention may be directed to human alpha and beta coronaviruses such as the human alpha coronaviruses referred to as HCoV-229E and HCoV-NL63 and the human beta coronaviruses referred to as HCoV-OC43, HCoV-HKLH, MERS-CoV, SARS-CoV and SARS-CoV-2. SARS-CoV-2 is severe acute respiratory syndrome coronavirus 2 which was originally identified in China at the end of 2019; it has also been referred to as 2019-nCoV and the disease it causes is referred to as COVID-19. The particles the present invention may potentially be able to exert antiviral activity against viruses which are not dependent on HSPG-binding to effect viral entry into the host cells. Recent studies have shown that sulfonated nanomaterials may have antiviral activity beyond HSPG-dependent viruses, for example against influenza virus or vesicular stomatitis virus [Cagno et al., Antimicrobial Agents and Chemotherapy, (2020) DOI: 10.1128 / AAC.02001-20],

[0100] Therefore, in some embodiments there is provided a pharmaceutical composition comprising a particle of the present invention or a pharmaceutically acceptable salt or salts thereof, or a pharmaceutical composition as defined herein for use in the prevention or treatment of viral infections associated with influenza (such as Influenza A H3N2 or H1N1 virus), adeno-associated virus (AAV), Newcastle disease virus (NDV), and vesicular stomatitis virus (VSV).

[0101] Throughout this specification, where mention is made of a human virus, the scope of the invention covers the same virus in non-human animal species. For example, reference to human immunodeficiency virus is intended to cover immunodeficiency viruses in other animals such as feline immunodeficiency virus or bovine immunodeficiency virus.

[0102] In some embodiments there is provided a pharmaceutical composition comprising a particle of the present invention or a pharmaceutically acceptable salt or salts thereof, or a pharmaceutical composition as defined herein, for use in the prevention or treatment of viral infections associated with one or more viruses selected from herpes simplex virus (HSV), herpes simplex virus 2 (HSV-2), human rhinovirus-8 (HRV-8), adenovirus, adeno-associated virus, human papillomavirus (HPV), respiratory syncytial virus (RSV), dengue virus, norovirus, lentivirus, human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), human metapneumovirus (HMPV), human parainfluenza virus type 3 (HPIV-3), coronavirus (such as MERS-CoV, SARS-CoV, or SARS-CoV-2), foot-and- mouth disease virus, hepatitis B virus, hepatitis C virus, Ebola virus, nipah virus, Rift Valley fever virus, West Nile virus, Crimean Congo virus, Toscana virus, ZIKA virus, Chickungunya virus (CHIKV), Akabane virus (AKAV) or Schmallenberg virus (SBV), influenza (such as Influenza A H3N2 or H1N1 virus), adeno-associated virus (AAV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV) or Human coronavirus OC43 (OC43).

[0103] In some embodiments there is provided a pharmaceutical composition comprising a particle of the present invention or a pharmaceutically acceptable salt or salts thereof, or a pharmaceutical composition as defined herein, for use in the prevention or treatment of viral infections associated with one or more viruses selected from herpes simplex virus (HSV), herpes simplex virus 2 (HSV-2), human rhinovirus-8 (HRV-8), Respiratory Syncytial Virus (RSV), human coronavirus (such as human corona virus OC43), SARS-CoV-2 and Cytomegalovirus (CMV).

[0104] In some embodiments, the subject in need of treatment according to the present invention as described herein, is a human. In other embodiments, the subject is an animal, such as a mammal, in need of such treatment.

[0105] Non-Therapeutic Uses

[0106] Also provided herein is a method of sterilisation or viral disinfection, comprising using an effective amount of the particle of the present invention or a composition comprising the particle of the present invention.

[0107] The method of sterilisation or viral disinfection may comprise the steps of:

[0108] (i) providing the particle or a plurality of particles of the present invention, or a salt or salts thereof;

[0109] (ii) contacting a surface contaminated by a virus, or suspected of being contaminated by a virus, with the particle or a plurality of particles of the present invention as provided in step (i).

[0110] The virus is may be one or more viruses selected from herpes simplex virus (HSV), herpes simplex virus 2 (HSV-2), human rhinovirus-8 (HRV-8), adenovirus, adeno- associated virus, human papillomavirus (HPV), respiratory syncytial virus (RSV), dengue virus, norovirus, lentivirus, human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), human metapneumovirus (HMPV), human parainfluenza virus type 3 (HPIV-3), coronavirus (such as MERS-CoV, SARS-CoV, or SARS-CoV- 2), foot-and- mouth disease virus, hepatitis B virus, hepatitis C virus, Ebola virus, nipah virus, Rift Valley fever virus, West Nile virus, Crimean Congo virus, Toscana virus, ZIKA virus, Chickungunya virus (CHIKV), Akabane virus (AKAV) or Schmallenberg virus (SBV), influenza (such as Influenza A H3N2 or H1N1 virus), adeno-associated virus (AAV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV) or Human coronavirus OC43 (OC43). For example, the virus is one or more virus selected from herpes simplex virus (HSV), herpes simplex virus 2 (HSV-2), human rhinovirus- 8 (HRV-8), Respiratory Syncytial Virus (RSV), human coronavirus (such as human corona virus OC43), SARS-CoV-2 and Cytomegalovirus (CMV).

[0111] The surface contaminated by a virus, or suspected of being contaminated by a virus, is found on medical equipment, furniture or clothing, or in medical rooms such as operating theatres or hospital wards.

[0112] There is also provided a device for sterilisation or viral disinfection comprising a composition as defined herein and means for dispensing the composition. The device may be a spray and the means may comprise a spray applicator.

[0113] The means may comprise a support material impregnated with the composition. For examples, the support material impregnated with the composition is a textile, a woven or non-woven fabric, an absorbent sheet or a sponge.

[0114] Brief Description of the Figures

[0115] Figure 1 shows A) Preparation of PSSx-PBzMAynanoparticles via polymerization- induced self-assembly and B) schematic representation of potential antiviral modes of action of PSSx-PBzMAynanoparticles, depicting a virustatic and virucidal mode of action.

[0116] Figure 2 shows Transmission Election Microscopy (TEM) Analysis of PSSx-PBzMAy Nanoparticles; 12:50 (Top), 47:50 (Middle), 47: 100 (Bottom).

[0117] Figure 3 shows Dose Response Assay A) HSV-2, B) RSV, C) CMV, D) HRV-8 and D) OC43. Each virus was treated with each antiviral for 1 hour prior to addition to Vero Cells and subsequent incubation at 37°C, 5% CO2 for 27-96 hours (virus dependant) until visible plaques form, prior to staining with 0.1% crystal violet solution and counting of viral plaques at 4X Objective.

[0118] Figure 4 shows a Virucidal Assay A) HSV-2, B) RSV, C) CMV, D) HRV-8, E) OC43. Each virus was treated with each antiviral for one hour prior to dilution subsequent incubation at 37°C for 24 -96 hours (virus dependant) until visible plaques form, prior to staining with 0.1% crystal violet solution and counting of viral plaques at 4X objective. Line indicates threshold for >2 log reduction (n=3)

[0119] Figure 5 shows FAIRY Assay with following treatment with 12:50, 47:50 and 47: 100 for one hour. The chemically treated samples were mixed with TO-PEG (final TO-PEG concentration 0.5 mM) and fluorescence recorded (XEX=5 10 nm and XEm=533 nm). a) HSV-2 treated at a final concentration of 40 pg / mL, B) RSV at a final concentration of 40 pg / mL, C) CMV at a final concentration of 100 pg / mL, D) OC43 at a final concentration of 50 pg / mL. Results presented as means ±SD. Statistical analysis were performed using one-way ANOVA with multiple comparisons (* * = p < 0.01, * * * = p< 0.001, ns = not significant) (n=3).

[0120] Figure 6 is a schematic representation of the in vivo intranasal dosing and lung harvest regime.

[0121] Figure 7 shows weight loss curves, relative to 0 days post infection (dpi) weights.

[0122] Figure 8 shows RSV viral lung titres, analysed using TCID50, in the supernatant collected from homogenised lung showing a 2-log (99%) and 4-log (99.99%) reduction in lung viral titres with virustatic 12:50 and virucidal material 47:50 respectively (1.5 dpi) and subsequent viral titre recovery (2-4 dpi).

[0123] Figure 9 Shows percentage RSV viral lung titre recovery relative to non-treatment control (NTC).

[0124] Figure 10 Shows RSV plaque assay performed on 4 dpi lung lysate. Examples

[0125] Poly(styrene sulfonate) (PSS)-functional block copolymer nanoparticles may be prepared by PISA. The particles were investigated in terms of their antiviral properties (Figure 1). PSS is well known to bind to a wide range of viruses giving the formed nanoparticles the potential for broad-spectrum efficacy

[0042] , Poly(styrene sulfonate)-6- poly(benzyl methacrylate) (PSSx-PBzMAy) nanoparticles for study were prepared via reversible addition fragmentation chain-transfer (RAFT) PISA in ethanol / water mixtures following a previously reported related protocol for poly(potassium 3- sulfopropyl methacrylate)functional PBzMA nanoparticles.

[0043]

[0126] Three PSSx-PBzMAydiblock copolymer nanoparticles with different copolymer compositions (PSS -PBzMAso, PSS47PBZMA50 and PSS47-PBZMA100) were prepared in order to investigate the effect of polymer block length on the antiviral properties of these nanoparticles. For brevity, each PSSx-PBzMAynanoparticle will be referred to herein by the degree of polymerization (DP) of each block. For example, PSS47- PBzMAso will be referred to as 47:50.

[0127] Characterisation of PSS-PBzMA Nanoparticles

[0128] The block copolymer nanoparticles were prepared to have differing block lengths but to have their overall diameter remain consistent.

[0129] Table 1 aMean hydrodynamic diameters obtained via DLS for nanoparticles synthesised in methanol-water mixtures. DLS polydispersity index values are indicated in brackets.bMean hydrodynamic diameters obtained via DLS for nanoparticles after a freezethaw cycle and then redispersion in water. DLS polydispersity index values are indicated in brackets.cNumber-average particle diameters determined from TEM images by analysing 200 particles using ImageJ software.dNaggrepresents mean copolymer aggregation number per particle and was calculated using Equation 1. Rcorewas determined from D„, xi0 / was assumed to be zero, and N chain was calculated using a PBzMA density of 1.15 g cm ', and the target copolymer composition. [44-46]eSQgg represents average number of copolymer chains per unit surface area of a particle and was calculated using Equation 2 where Rcorewas determined from D„.[45, 46]

[0130] The prepared nanoparticles had well-defined spherical morphologies and numberaverage diameters of ~200 nm, as determined by transmission electron microscopy (Figure 2, Table 1). The mean intensity average hydrodynamic diameter of the as- prepared nanoparticles was ~230 nm (Table 1). In order to transfer the nanoparticles into an aqueous medium for further study they were lyophilized and re-dispersed in water. This is vital for further study as an antiviral, as initial synthesis was conducted under non-sterile, alcoholic conditions. Upon re-dispersion, the measured particle diameter remained consistent (Table 1).

[0131] Whether PSS-decorated nanoparticle surfaces could confer a material with antiviral activity was assessed, along with whether this activity irreversibly destroys the virus upon contact.

[0132] Biocompatibility Assessment

[0133] To investigate antiviral activity of the synthesized PSSx-PBzMAynanoparticles in vitro, low levels of cytotoxicity need to be established so that cell monolayers (required for viral plaque assays) can be grown and remain viable post treatment. A MTT assay, using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, was performed to assess cell metabolic activity. Here, through enzymatic reduction of the MTT reagent, by mitochondrial succinate dehydrogenase to the insoluble formazan product, an indication of cell health can be established. This assay has several advantages as it is sensitive, quantitative and provides a linear correlation between cell viability and absorbance. Vero cells (ATCC (CCL-81)) were grown to 95-100% confluency in a 96 well plate, prior to addition of PSSx-PBzMAynanoparticles at concentrations ranging from 0-800 / zg / mL for 24 hours. The MTT reagent was added and incubated at 37°C for 4 hours prior to measurement of the insoluble formazan product by recording absorbance at 490 nm (ESI Figure S I). All three antivirals tested displayed excellent biocompatibility, even at 800 / zg / mL, with no statistically significant difference from 100% viability control.

[0134] This observation is consolidated through visual inspection of crystal violet stained monolayers post treatment, which demonstrated no change in morphology or change in growth pattern. The finding of such promising biocompatibility is to be noted, as there are currently only a few examples of polymer nanoparticles synthesised via PISA which display comparable levels of biocompatibility [47, 48] ,

[0135] Broad-Spectrum Antiviral Activity of PSS-PBzMA Nanoparticles

[0136] Following establishing biocompatibility, antiviral activity against Herpes Simplex Virus 2 (HSV-2), Respiratory Syncytial Virus (RSV) and Cytomegalovirus (CMV) were established through determining half-maximal inhibitory concentration (IC50).

[0137] These values provide a comparison point between different antivirals in terms of efficacy (Figure 3D).

[0138] IC50 and IC90 values were calculated through non-linear regression analysis (Table 2). All three nanoparticles demonstrated antiviral activity against a range of viruses, with IC50 values < 1 zg / mL for HSV-2, < 8 zg / mL for RSV, and between 10 - 400 zg / mL for CMV.

[0139] Table 2 These nanoparticles are very similar in overall diameter (Table 1), suggesting that there must be another factor conferring the variation of antiviral efficacy between different nanoparticle architectures. The number of copolymer chains present in a nanoparticle can be represented by the mean aggregation number (Nagg) and calculated using Equation 1, [44-46]

[0140] Equation 1 where xi0 / is the fraction of solvent in the core of the nanoparticle, Rcoreis the radius of the nanoparticle core, and N chain is the volume occupied by a single copolymer chain in the core of nanoparticle. Rcorewas determined from D„, xi0 / was assumed to be zero, and N chain was calculated using a PBzMA density of 1.15 g cm '. and the target copolymer composition. This can be extended to determine the average number of copolymer chains per unit surface area of a nanoparticle (Sogg), using Equation 2.

[0141] Equation 2

[0142] For the PSSx-PBzMAynanoparticles studied herein Naggand Saggwere calculated assuming xJ0; = 0, due to the particles being lyophilized and redispersed, with minimal changes to D*, and previous small-angle X-ray scattering (SAXS) analysis of closely related particles

[0040] , Rcorewas determined using the diameter obtained from TEM image analysis and N chain was calculated using a PBzMA density of 1.15 g cm ' and the target copolymer composition (Table 1).

[0045] The calculated values of Naggand Saggbegin to establish a structural-property relationship to antiviral efficacy.

[0143] The variation in antiviral efficacy observed in Figure 3 is not only dictated by the degree of polymerization of PSS, but also by the distribution and density of PSS chains on the nanoparticle surface. All three antivirals were effective against all viruses assessed via dose-response assays, suggesting that once the PSS block length is greater than or equal to 12 (in this work) the interaction with a virus is sufficient enough to inhibit infection. It should be noted that smaller PSS block lengths may also have antiviral effects. On increasing the degree of polymerization of the PSS corona DP from 12 to 47, a ten-fold decrease in IC50 was observed with HSV-2, from 0.922 / zg / mL to 0.095 / zg / mL. The increase of degree of polymerization of the corona from 12 to 47, despite both Naggand Saggremaining consistent, resulted in antiviral efficacy improving across all viruses assessed.

[0144] The observed improvement of antiviral efficacy following the increase of corona block DP suggests that increasing the number of sulfonated groups present on the corona, thus increasing the number of anionic groups on the nanoparticles, improves antiviral efficacy. In contrast, when the PBzMA core degree of polymerization doubles from 50 to 100, but the PSS corona DP remains the same, the IC50 value against HSV-2 increases nearly four-fold from 0.095 zg / mL or 0.372 zg / mL. The doubling of core size also results in a decrease of both Naggand Saggvalues, indicating fewer copolymer chains present in a given nanoparticle and, perhaps more importantly, fewer PSS chains present per unit surface area of a given nanoparticle. The observed increase of IC50 values following increase of PBzMA core size observed indicating decreased antiviral efficacy, across all viruses tested, demonstrates that a higher density of PSS chains on the nanoparticle confers a more potent antiviral. The IC50 values of all of the nanoparticles from this study remained low with the most effective nanoparticle conformation produced in this study being 47:50.

[0145] PSS-PBzMA Nanoparticles Demonstrate Extracellular Virucidal Mode of Action

[0146] Following the establishment of antiviral activity against a range of viruses, it was imperative to establish whether the nanoparticles were either virustatic or virucidal. As previously discussed, virustatic materials make contact with the virus and prevent viral infection of cells via a reversible process. Changes in pH, temperature or dilution can result in the disassociation of the antiviral from the virus, leaving an intact, infectious virion. Conversely, virucidal materials make direct contact with the viral particle and irreversibly destroys the virus upon contact, rendering it inert and unable to infect. This allows a virucidal assay, which uses serial dilution across a 96-well plate, to be used to determine mode of action. In this instance, each antiviral was mixed with each virus for 60 minutes before being serially diluted. If the interactions between the antiviral and virus were reversible (nondestructive / virustatic), then serial dilution would induce dissociation of the antiviral from the viral surface and viral plaques would be observed at high levels of dilution (Figure 4). Here, when the reduction of plaque forming units per millilitre (PFU / mL) following the assay are >2 log lower than the NTC, antivirals are considered to be virucidal. Anything less than a 2 log reduction is deemed virustatic. It is imperative when conducting the virucidal assay that some plaques are observed (i.e. the antiviral treated sample has a value greater than zero) to ensure the assay has been performed correctly.

[0147] Following the virucidal assay, it was clear that 12:50 shows a virustatic mode of action against all viruses, evidenced by a 0.588-log reduction in PFU / mL at a concentration of 100 zg / mL with HSV-2 for example (Figure 4A). However, we observe virucidal activity with two of the antivirals tested, 47:50 and 47:100, indicated by a>2 log reduction in PFU / mL post treatment. This indicates that an increase in the DP of the PSS from 12 to 50 allows a virucidal mechanism to be achieved. The same shift in mechanism of action from virustatic to virucidal following increase of PSS DP was also observed with RSV, CMV, HRV-8 and OC43 (Figure 4 B-E). Comparing the efficacy of both virucidal candidates, 47:50 and 47:100, there is no change in mode of action when increasing core-size. However, lower IC90 values are observed with a larger core against HSV-2 and RSV, indicating higher potency.

[0148] The above virucidal assay, which is the current gold-standard approach to determining virucidal mechanism

[0049] , clearly indicated a difference in mechanism between the synthesised nanoparticles. In order to further support this finding, cell-free genome exposure assays

[0023] were conducted using 47:50 and 47:100. The assay confirms the destruction of HSV-2 virions post-virucide treatment and subsequent release of intact viral genome in a cell free environment through inclusion of a DNase treatment step. If the viral genome is released following treatment (as would be the case for a virucide), the genome would be accessible to enzymatic degradation by the DNase. The degradation of genome results in virus specific primers being unable to bind onto their target sequence, resulting in no amplification or detection by qPCR. Virions left intact after antiviral treatment will have their genome intact, resulting in amplification and detection of virions. Initial results were inconclusive, with only minor fold changes being observed, relative to the NTC (ESI Figure S3 A). Highly sulfonated polymers have previously been reported to inhibit enzymatic activity [50, 51], which could explain the inconclusive findings from this study (ESI Figure S3 A). In order to probe if the polymer nanoparticles were inhibiting DNase activity, 47:100 was added to a previously extracted, untreated HSV-2 genome followed by DNase treatment and qPCR (ESI Figure S3B). In the event that the antiviral did not inhibit DNase activity, a high cycle threshold (cT) value would be expected. Whereas if the antiviral inhibited the DNase, no degradation of DNA would occur, and so a low cT value would be recorded. In the sample containing 47:100, a low cT value is observed with a statistically significant difference to the sample containing no 47:100, confirming that it was inhibiting the DNase enzyme. This means that this cell-free genome-exposure assay cannot be used to determine virucidal mechanism with these nanoparticles, meaning another assay was needed.

[0149] A cell-free fluorescence-based assay developed in-house termed Fluorescence Assay for vIRal Integrity (FAIRY), was utilised to determine the mode of antiviral activity. In FAIRY, virucides are identified using a dye-polymer conjugate that has a ’turn-on’ fluorescence only upon virion destruction. FAIRY was conducted with HSV-2 following treatment with each antiviral for 1 hour at a final concentration of 40 zg / mL. Treatment of HSV-2 with 47:50 and 47:100 had previously been observed to be virucidal in the cell-based assay, whilst 12:50 had only demonstrated virustatic activity.

[0150] Statistically significant increases of fluorescence, relative to the NTC, are only observed following HSV-2 treatment with 47:50 and 47:100. Conversely, following treatment with 12:50, fluorescence remains the same with no statistically significant difference to the NTC (Figure 5A). Moreover, the nanoparticles on their own at the same final concentration have no impact on the assay, with intensity values relative to the blank (MilliQ Water) showing no statistically significant difference (ESI Figure S4), addressing the limitations of the Genome Exposure Assay. FAIRY was able to differentiate between virustatic and virucidal antivirals, supporting observations made by dilution assay.

[0151] These findings correlate with gold standard method alongside initial development studies

[0052] and thus further shows that FAIRY is a powerful correlative method for determining mode of action. Subsequent experiments were then performed with FAIRY to determine if the same shift from a virustatic to a virucidal mode of action on increase of degree of polymerization of PSS was observed with a broader range of viruses. Observations made with HSV-2 were consolidated following investigation with respiratory syncytial virus (RSV), Cytomegalovirus (CMV) and Human coronavirus OC43 (Figure 5 B-D). The FAIRY assay has allowed the scope of viruses investigated to be broadened, including those linked to community health concerns such as OC43 and RSV. The confirmation of a virucidal mode of action against a broad-spectrum of enveloped viruses, alongside excellent compatibility, broadens the potential scope of applications of this class of block-copolymer nanoparticles with HSPG mimicking PSS corona.

[0152] The inventors have utilised PISA to form block co-polymer nanoparticles of ~200 nm with a hydrophobic PBzMA core and hydrophilic PSS corona. A range of nanoparticles with varying core and corona lengths were synthesised, while maintaining the overall diameter, to explore the antiviral properties and mode of action. These nanoparticles are shown to be non-toxic to vero cells at a concentration of 800 / zg / mL, with such promising levels of biocompatibility previously being rarely reported. Dose response assays against HSV-2, RSV and CMV show that each nanoparticle composition is antiviral, IC50 values < 1 / zg / mL for HSV-2, < 8 zg / mL for RSV, and between 10 - 400zg / mL for CMV. The colloids with the smallest core and shell were antiviral but with a virustatic mechanism. Upon increasing the corona size (but retaining the core size) a virucidal mechanism is achieved. This is then maintained upon further increasing the core size and confirmed using plaque and fluorescent based assays. The shift from a virustatic to a virucidal mode of action demonstrated with HSV-2 upon increasing corona size was further observed on a wider range of enveloped viruses (respiratory syncytial virus (RSV), Cytomegalovirus (CMV) and Human coronavirus (OC43). The difference in potency is attributed to the differences in PSS chain density on the surface of the nanoparticles.

[0153] These readily synthesised polymer nanoparticles are promising antivirals and further enhance our understanding of the properties required to achieve a virucidal mechanism in biocompatible extracellular antivirals. Methods

[0154] Synthesis and Characterisation of PSS-PBzMA Nanoparticles

[0155] Materials: 4-Vinylbenzenesulfonic acid sodium salt (SS, 98%) and 4,4’- azobis(4cyanovaleric acid) (ACVA, 99%) were purchased from Sigma-Aldrich (UK) and both chemicals were used as received. Benzyl methacrylate (98%) was purchased from Alfa Aesar (UK) and purified by passing through a column of activated basic alumina before use. 4-Cyano-4-(2-phenylethane sulfanylthiocarbonyl) sulfanylpentanoic acid (PETTC) was prepared in-house using previously published methods

[0053] , Deuterium oxide (D2O) and methanol (>99.9%) were purchased from Cambridge Isotope Uaboratories (UK) and Fisher Scientific (UK), respectively, and used as received. Deionized water was generated by an Elga Purelab Option water purification system.

[0156] Preparation of PSS via RAFT solution polymerization: A typical protocol for the synthesis of a PSS macro-CTA via RAFT solution polymerisation at 15% solids was as follows. For PSS47 macro-CTA, SS (7.26 g, 35.2 mmol), PETTC (239. 1 mg, 0.7 mmol, dissolved in dioxane), ACVA (39.5 mg, 140.8 mmol, PETTC / ACVA molar ratio = 5), and pH 5.5 acetate buffer (42.5 g, final buffer / dioxane ratio = 3) were charged into a 100 mL a round-bottomed flask equipped with a nitrogen inlet.

[0157] The sealed flask was deoxygenated via purging nitrogen at ambient temperature for 30 min. After deoxygenation, the round-bottomed flask was immersed into a preheated water bath at 70 °C for 180 min. The resulting crude PSS47 macro-CTA (96% conversion; Mn= 6000 g mol1, Mw / Mn= 1.05) was purified via dialysis against 10: 1 water / methanol and further dried under vacuum overnight. The degree of polymerization was calculated using1H NMR spectroscopy by comparing the integrated proton signals corresponding to the polymer backbone at 0.4-3. 1 ppm with those corresponding to the CH2 protons of the PETTC chain end at 3.3-3.7 ppm.

[0158] Preparation of PSS-PBzMA diblock copolymer nanoparticles: A typical protocol for the synthesis of PSSx PBzMAydiblock copolymer nanoparticles at 10% w / w solids in methanol / water mixtures was as follows. For PSS12- PBzMAso synthesised in methanol / water at 10% w / w methanol, PSS12 macro-CTA (469.6 mg, 0.185 mmol), BzMA (1630.4 mg, 9.252 mmol), ACVA (17.3 mg, 0.062 mmol, CTA / initiator molar ratio = 3) and methanol / water (18.9 g) were charged into a 50 mL round-bottomed flask equipped with a nitrogen inlet. The sealed flask was deoxygenated via purging nitrogen at ambient temperature for 10 min. After deoxygenation, the round-bottomed flask was immersed into a preheated water bath at 70 °C for 24 h to ensure complete conversion of BzMA. The polymerizations were quenched by cooling to room temperature and exposing to air. Subsequent polymerizations were performed by varying the target copolymer composition, using the procedure described above.

[0159] Characterization: Proton (1H) NMR spectra were acquired using a 400 MHz Bruker Advance III spectrometer with 128 scans being averaged per spectrum. Samples were dissolved in D2O prior to NMR analysis. PSS molar mass distributions were determined by aqueous gel permeation chromatography (GPC) using a GPC equipped with two PL aquageTOH MIXED-H 8 zm columns at ambient temperature. Phosphate buffer with 30% v / v methanol at pH 9 was as used as an eluent at a flow rate of 1.0 m / min-1. A refractive index detector (Shodex RI-101) was used and the system was calibrated with a series of near-monodisperse poly(ethylene oxide) standards. Transmission electron microscopy (TEM) images were recorded using a FEI Tecnai G2 20 instrument operating at an accelerating voltage of 200 kV and connected to a Gatan Ik CCD camera. Samples for TEM observation were prepared by depositing 2 zL of diluted samples (approximately 0.1% w / w) onto 400 mesh carbon-coated copper grids. The samples were stained in a vapor space above ruthenium tetroxide (RuCL) solution for 7 min at ambient temperature. The mean nanoparticle diameters were determined using Image J software, and over 200 randomly selected particles were measured for each sample. Dynamic Light Scattering (DLS) studies were performed using a Malvern Zetasizer Nano ZS instrument equipped with a He-Ne solid-state laser operating at 633 nm and backscattered light at a scattering angle of 173°. Nanoparticle dispersions were diluted to approximately 0.1% w / w using deionized water. Samples were analyzed at 25 °C using disposable plastic cuvettes, and data were averaged over three consecutive measurements. Methanol-containing nanoparticle dispersions were lyophilzed and subsequently dispersed in sterile phosphate buffered saline (PBS) prior to assays being performed.

[0160] Virus and Cell Culture

[0161] Herpes-Simplex Virus 2 (HSV-2), Respiratory Syncytial Virus (RSV), Cytomegalovirus (CMV) and Coronavirus (OC43) stocks were initially isolated and verified from clinical samples and then kindly donated by Professor Pamela Vallely (University of Manchester School of Medical Sciences). Additional stocks of HSV-2, RSV and CMV were grown in Vero cells and stored at -80°C. Vero cells at a confluency of 80% were infected with HSV-2 stock and incubated until 90% CPE was observed. Additional Coronavirus OC43 stocks were grown on Mv 1 Lu cells in-lab and stored at - 80 °C.

[0162] Cell culture was conducted under asceptic conditions in a class II microbiological safety cabinet (MBSC-II). Vero cells (ATCC (CCL-81)) were kindly donated by Mr David Dennington (Faculty of Biology, Medicine and Health, University of Manchester). Vero cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) modified with high glucose, L-glutamine, phenol red, and sodium pyruvate (Thermo Fisher Scientific, Loughborough, UK). This media was further supplemented with 1% penicillin / streptomycin (P / S) (Merck Life Science UK Ltd., Dorset, United Kingdom) and 10% heat inactivated fetal bovine serum (FBS) (Merck Life Science UK). Where cell culture relating to FAIRY is concerned, Vero cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) modified with high glucose, L-glutamine, no-phenol red, and sodium pyruvate (Thermo Fisher Scientific, Loughborough, UK). Media was supplemented with 1% penicillin / streptomycin (P / S) only (Merck Life Science UK Ltd., Dorset, United Kingdom). Mv 1 Lu cells (mink lung epithelial cells) were kindly donated by University of Manchester School of Biological Sciences (Dr. Pamela Vallely) and were maintained in DMEM modified with a Phenol-Red Free, High- Glucose DMEM supplemented with 1% P / S. Cells were cultivated at 37°C with 5% CO2 in 75 cm2flasks and passaged in a 1 :6 ratio when confluent.

[0163] Dose Response Assay with HSV-2, RSV and CMV

[0164] Vero cells were seeded into 24 well-plates at a concentration of 175 000 cells / mL at a final volume of 500 zL per well. Cells were incubated until a confluency of 90-95% was observed for HSV-2 assays, and 70-80% for RSV and CMV assays. Sterile eppendorfs were prepared with concentrations of antiviral solution ranging between 0 - 400 zg / mL in sterile PBS to a final volume of 570 zL. A 1 : 100 dilution of isolated virus stock was made with DMEM and the original isolated stock; 30 zL of this stock was added to each eppendorf that contained antiviral and incubated for 1 hour at 37 °C and 5% CO2. One eppendorf just containing PBS was also incubated as a no virus control. Media was removed from the 24 well plate and 200 zL of each antiviral:virus mixture was added to each well and incubated at 37°C and 5% CO2 for 1 hour. The antiviralvirus mixtures were removed from the plate and replaced with 500 zL of 20% methylcellulose in DMEM (supplemented with 2% FBS) and incubated until visible viral plaques were formed (24 - 72 hours, dependant on virus).

[0165] Methylcellulose DMEM was then removed and the wells were stained with a 0.1% crystal violet solution. Plaques were then counted at 4X objective on an optical microscope.

[0166] Virucidal Assay with HSV-2

[0167] Vero cells were seeded into a 96-well plate at a concentration of 150 000 cells / mb at a final volume of 100 zL per well and incubated until 90-95% confluency was observed. A 1 : 1 dilution of virus stock and antiviral solution to a final volume of 110 zL was created (a 1 :2 antiviral dilution). A no treatment control (NTC) was also created with 55 zL of virus stock mixed with 55 zL of PBS. Both virus plus antiviral and NTCs were incubated for 1 hour at 37°C. Six Eppendorf tubes were prepared, each containing 450zL of cell culture medium (three were used with the antiviral and three with the NTC). After incubation, 50 zL was taken from the virus-antiviral mix and added into the first Eppendorf tube, diluting this antiviral by a further 1 : 10. 50 zL was then serially diluted into the other two tubes, leaving three Eppendorfs at a 1:20, 1 :200, and 1 :2000 dilution of antiviral, respectively. This process was also repeated for the NTC. 50 zL from the virus plus antiviral mix (1 :20 dilution) was then added to wells Al and A2 of the 96- well plate. 50 zL from the 1:200 virus-antiviral mix was added to wells A3 and A4, and 50 zL from the 1 :2000 virus-antiviral mix was added to wells A5 and A6. 50 zL from these wells was then serially diluted down the plate to row G. In well Hl, 50 zL of the originally incubated virus plus antiviral stock was added, resuspended, and serially diluted 1 :3 to well H6. This process was repeated for the NTC in wells A7-A12. The plate was then incubated for 1 hour at 37°C and 5%CC>2.

[0168] Virus: antiviral mix was removed and replaced with 100 zL of methylcellulose DMEM. The plate was then incubated at 37°C and 5%CC>2 until visible plaques were formed. Methylcellulose DMEM was removed and stained with 0.1% crystal violet solution.

[0169] Plaques were counted 4X objective on an optical microscope. MTT Assay

[0170] Vero cells were seeded onto a 96 well plate at a concentration of 150 000 cells / mL at a total concentration of 100 zL and incubated until 90-95% confluent. 50 zL of cell culture media was removed and 50 zL of antiviral was added resulting in final concentrations ranging between 0 - 800 zg / mL and incubated for 24 hours at 37°C and 5%CC>2. Alongside the antiviral, a control with PBS and a control with cell culture media were set up. 20 zL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay Reagent (Promega, Hampshire) was added and incubated for 4 hours at 37°C and 5%CO2. Absorbance at 490 nm was recorded and all results were normalised with the control that has not been treated with antiviral. Alongside this experiment, a control with Vero cells, antiviral and no assay reagent was performed.

[0171] Genome Exposure Assay

[0172] Enzymatic Treatment: Viral stock was mixed with antiviral at a concentration of 80 / zg / mL in a 1 : 1 ratio (final concentration 40 / zg / mL) alongside a ’no treatment control’ (NTC) consisting of a 1 : 1 volume ratio of viral stock and sterile deionised water, and then incubated at 35°C, 5 % CO2, for one hour. 10X TURBO ™ DNase was added to each sample in a ratio of 1 zg / mL of TURBO™ DNase per 1 / zg of DNA present in the most highly concentrated sample. An equal volume of TURBO™ DNase was added to all other samples prior to incubation at 35 °C, 5 % CO2 for thirty minutes. To deactivate the DNase, EDTA was added to samples at a final concentration of 15 mM and heated at 75 °C for ten minutes.

[0173] Viral Genome Extraction: Genomes were extracted using the Purelink Viral RNA / DNA Minikit (ThermoFischer Scientific, UK) according to manufacturer’s instructions. Purified viral genome was stored at -80 °C.

[0174] Real-Time Polymerase Chain Reaction (qPCR) Amplification: qPCR was performed with the PowerUp™ SYBR Green™ Master Mix (Applied Biosystems). See Table 2 for primers used (Eurofins, Europe). Samples were made up to 20 zL according to manufacturer’s instructions, amplification and detection were conducted by qPCR using Applied Systems StepOne Plus™ machine and associated software (ThermoFischer Scientific, UK).

[0175] Table 2 Primers Used in qPCR Studies

[0176] Fluorescence Assay for vIRal Integrity (FAIRY)

[0177] First thaw HSV-2, RSV CMV and OC43 stocks (with no phenol red or FBS) was added in a 1 : 1 ratio to 80 zg / mL antiviral (final concentration 40 / zg / mL)and incubated for 1 hour at 37 °C. For CMV and OC43, final concentrations of 100 zg / mL and 50 zg / mU were used respectively. Following treatment 50 zL of TO-PEG (0.5 mM) and 50 zL of the chemically treated samples (and non-treated samples) were added to a black 96-well flat bottom plate. Samples were incubated in the dark for 10 minutes and the fluorescence measured (2)EX=5 10 nm and 2)Em=533 nm).

[0178] In Vivo Studies

[0179] Intranasal RSV Infection and Antiviral Colloid Dosing

[0180] Mice (C57BL / 6, Charles river) were housed at an AALAC International -accredited institution and ethically cared for in accordance with The Guide for the Care and Use of Laboratory Animals.

[0181] Eight week old female C5BL / 6 mice were anaesthetized by inhalation of isoflurane and infected intranasally with 1.5x104 PFU of RSV in a volume of 30 pL in PBS. On Day 1 post infection, mice were intranasally dosed with 25 pg of either PSS -PBzMAso (12:50, n=12) or PSS47-PBZMA50 (47:50, n=12) or PBS sham (NTC, n=12) in a volume of 25 pL. Daily weights were monitored until culling. Figure 7 shows weight loss curves relative to 0 days post infection (dpi) weights.

[0182] Mice were culled on Days 2-4 post RSV infection and lungs harvested. All five lung lobes were collected into 750 pL sterile PBS in 1.5 mL RING homogenizer tubes with a pre-loaded stainless steel bead. Lung tissues were homogenized by bead-milling (2x times for 4 minutes on high speed), or until homogenate does not contain tissue chunks.

[0183] Lung lysate was then used in TCID50 assays.

[0184] Figure 6 shows a schematic representation of this process of in vivo intranasal dosing and lung harvest regime.

[0185] Results from the in vivo studies are presented in Figures 7 to 10. Data are presented as means ±SEM of at least 4 animals per experimental group and are representative of two independent experiments.

[0186] Median Tissue Culture Infectious Dose (TCID50) Assay

[0187] Vero cells were seeded onto a 96-well plate at a density of 8 000 cells / well at a final volume of 100 pL and incubated for 2 hours at 37°C, 5% CO2 to allow cells to adhere. 80 pL of media was added to wells A-Dl, bringing volume to 180 pL. 20 pL of murine lung lysate was added to A-Dl (bringing the total volume to 200 p). 100 pl of this mix was then transferred to the next four wells (A2-D2). This 1 :2 serial dilution was repeated across the plate, with the exception of wells A12-D12, which were left unexposed to sample as a negative control. Plates were then incubated for 3 days at 37°C, 5% CO2 to allow for virus to infect cells. Virus titre was determined by cytopathic effect observed in wells via observation of cell monolayers at 10X objective. TCID50 values were calculated using the Spearman-K Naber method.

[0188] Figure 8 shows RSV viral lung titres, analysed using TCID50, in the supernatant collected from homogenised lung showing a 2-log (99%) and 4-log (99.99%) reduction in lung viral titres with virustatic 12:50 and virucidal material 47:50 respectively (1.5 dpi) and subsequent viral titre recovery (2-4 dpi). Figure 9 Shows percentage RSV viral lung titre recovery relative to NTC. Figure 10 Shows a RSV plaque assay performed on 4 dpi lung lysate. These results confirm both that particle according to the present invention have efficacy in vivo and that, in vivo, 12:50 has a virustatic mode of action whilst 47:50 has a virucidal mode of action.

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Claims

CLAIMS1. A particle formed from one or more block co-polymers having the structure:L-[A]x-Z>-[B]y-LWherein:A is a monomer residue comprising at least one sulfonate or sulfate substituent,B is a hydrophobic monomer residue,L is a capping group, x is greater than 3, and y is 25 to 200.

2. The particle of claim 1 wherein A has the following structure:Wherein:R1is aryl, Ci-2oalkyl, Ci-2oalkylene-aryl, C(O)OR4, C(O)NHR4, OC(O)R4, NHC(O)R4or sulfonate, andR2and R3are independently selected from hydrogen and C1-4 alkyl; and R4is Ci-10 alkyl or aryl; and whereinEach of the aryl, Ci-2oalkyl, Ci-2oalkylene-aryl, or R4groups is substituted with one or more sulfonate or sulfate groups, and is optionally substituted with one or more substituents selected from hydroxy, halo, Ci-4alkyl, Ci- 4alkoxy, aryl and cyano.

3. The particle of claim 2, wherein R1is selected from:Wherein K is Ci-nalkylene.

4. The particle of any one of claims 1 to 3 wherein A is a monomer residue derived from polymerisation of styrene sulfonate (SS).

5. The particle of any one of claims 1 to 4, wherein B is selected from a monomer residue having at least one substituent selected from aryl, branched or linear alkyl, branched or linear allyl, fluorinated alkyl, fluorinated aryl.

6. The particle of any one of claims 1 to 4 wherein B is selected from a monomer residue derived from polymerisation of styrenic compounds, methacrylic compounds, acrylamide compounds, fluorinated compounds, or compounds having a Ci-Cioalkyl group.

7. The particle of any one of claims 1 to 4 wherein B is selected from a monomer residue derived from polymerisation of benzyl methacrylate, methyl methacrylate, glycerol methacrylate, ethyl methacrylate, 2-ethylhexyl methacrylate, propyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, hexyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate, 2,2,2-trifluoroethyl methacrylate, lauryl methacrylate, allyl methacrylate, octyl methacrylate, t-butyl methacrylate, tert-butyl acrylate, diacetone acrylamide, lactic acid, dimethyl siloxane, styrene, or combinations thereof.

8. The particle of any one of claims 1 to 4 wherein B is a monomer residue derived from polymerisation of benzyl methacrylate, styrene, glycerol methacrylate, diacetone acrylamide, methyl methacrylate, tert-butyl acrylate or combinations thereof.

9. The particle of any one of claims 1 to 8 wherein the block co-polymer has the structure :L-(poly (styrene sulfonate))x-6-(poly(benzylmethacrylate))y-L10. The particle of any one of claims 1 to 9, wherein x is greater than or equal to 12.1 1. The particle of any one of claims 1 to 10, wherein x is from 5 to 100, from 12 to70, or from 40 to 60.

12. The particle of any one of claims 1 to 1 1, wherein y is from 25 to 75.

13. The particle of any one of claims 1 to 12, wherein the particle has a number average diameter of from 100 to 400 nm.

14. The particle of any one of claims 1 to 13, wherein each L group may be independently selected from hydrogen, hydroxyl, bromo, chloro, alkyl or one of the following groups:Wherein:R5is hydrogen, halo, cyano, CO2H, Ci-shaloalkyl, Ci-salkylene-OH, or Ci- 3alkylene-NH2;R6and R7are independently selected from hydrogen, and Ci-3alkyl optionally substituted with cyano, halo or CO2H;R8is S-Ci -isalkyl, S-aryl, NR9R10 or aryl, said Ci-isalkyl any aryl groups being optionally substituted with one or more substituents selected from Ci-3alkyl, hydroxy, halo, cyano, CO2H, and Ci-3haloalkyl;R9is hydrogen or Ci-3alkyl; andR10is hydrogen, C1-12 or aryl, said C1-12 alkyl and aryl groups being optionally substituted with one or more substituents selected from hydroxy, Ci-3alkyl, halo, cyano, CO2H, and Ci-3haloalkyl.

15. The particle of any one of claims 1 to 14, wherein each L group is independently selected from hydrogen, hydroxyl, bromo, chloro, or one of the following16. A composition comprising a plurality of particles according to any one of claims 1-15.

17. A method of sterilisation or viral disinfection, comprising using an effective amount of the composition of claim 16.

18. A device for sterilisation or viral disinfection comprising the composition of claim 16 and a means for dispensing the composition.

19. A pharmaceutical composition comprising a plurality of particles of any one of claims 1-15 and one or more pharmaceutically acceptable excipients.

20. The particle of any one of claims 1-15 or the pharmaceutical composition of claim 18 for use in the prevention or treatment of viral infections.

21. The particle or the pharmaceutical composition for use in the prevention or treatment of viral infections of claim 20, wherein the viral infection is herpes simplex virus (HSV), herpes simplex virus 2 (HSV-2), human rhinovirus-8 (HRV-8), adenovirus, adeno-associated virus, human papillomavirus (HPV), respiratory syncytial virus (RSV), dengue virus, norovirus, lentivirus, human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), human metapneumovirus (HMPV), human parainfluenza virus type 3 (HPIV-3), coronavirus (such as MERS-CoV, SARS-CoV, or SARS-CoV-2), foot-and- mouth disease virus, hepatitis B virus, hepatitis C virus, Ebola virus, nipah virus, Rift Valley fever virus, West Nile virus, Crimean Congo virus, Toscana virus, ZIKA virus, Chickungunya virus (CHIKV), Akabane virus (AKAV) or Schmallenberg virus (SBV), influenza (such as Influenza A H3N2 or H1N1 virus), adeno-associated virus (AAV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV) or Human coronavirus OC43 (OC43).

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