Virus model nanoparticle and uses thereof

Abstract

The present invention refers to a nanoparticle comprising a core of a metal or oxide metal coated with an organic compound, wherein said nanoparticle has a diameter and surface characteristics similar to a virus. The present invention also refers to methods and uses of the nanoparticle for determining the permeability of a membrane such as mucosal membranes, and the screening of active agents for the prevention of an infection caused by a virus, without the need to use real viruses.

Description

[0001]Virus model nanoparticle and uses thereof The invention relates to a virus model nanoparticle and its uses for the determination of the permeability of a membrane, and the screening of active agents for the treatment and / or prevention of an infection caused by a virus. BACKGROUND ART The spread of pathogenic viruses has become one of the main concerns due to the recent pandemic situation such as those caused by respiratory viruses like SARS- CoV-2 (severe acute respiratory syndrome coronavirus-2). This kind of viruses are entities that evolve quickly, and despite the efforts to produce new vaccines that can control them, their mutation rate give rise to situation in which viruses can evade the immune response of the infected hosts. Therefore, there is a continuous effort to study the pathogenic processes of viruses and develop new strategies for the prevention of virus infections, not only base on the vaccine investigation, but also on the production of new drugs that may cure or prevent the virus infection. In the process of studying pathogenic processes of virus infection and development of new therapeutic strategies in medicine, it is necessary to perform in vitro and in vivo assays with viruses per se in order to understand their pathogenic mechanisms and how they can be blocked by new drugs or vaccines. However, there are some risks associated with conducting experiments with pathogenic viruses such as the SARS-CoV-2, and high-level containment measures are needed, wherein said measures are only available in few laboratories (Xia, H., & Yuan, Z. (2022). Journal of Biosafety and Biosecurity, 4(1), 1-4). Therefore, there is a need to provide means for the study of pathogenic processes of new outbreaking viruses and the research on new therapeutic strategies against them. In the prior art there is a provision of synthetic in vitro models such as nanoparticles that mimic the size and shape of viruses that can be used in the research field. Gold nanoparticles coated with polyvinylpyrrolidone, having a size ranging from 60 to 140 nm, have been used as synthetic virus model for the study of host-pathogen early interaction 3D in vitro models of upper respiratory tract (Pennarossa, G., et al. (2021). Next Generation Culture Platforms for Reliable In Vitro Models: Methods and Protocols, 131-138). Multibranched spike bearing gold nanoparticles of 90 nm can be used as virus models in nebulization experiments in order to the person-to-person spread of the infection by aerosols  (Singh, A. et al. (2023). Science of The Total Environment, 860, 160503). However, in the case of respiratory viruses like those belonging to the family Coronaviridae, e.g. SARS-CoV-2, one of the most important aspects to study is their permeation through biological membranes like mucosal membranes. During this process, not only the size is an important factor to take into account but also the surface characteristics like the cationic and hydrophobic nature of viruses that enable them to interact and penetrate through mucosal membranes like oral or nasal membranes. Therefore, there is a need in the prior art to provide synthetic virus models or virus like nanoparticles that mimic not only the size feature but also the surface characteristics of a virus that allow them to interact and permeate through biological membranes. Thus, synthetic virus models that mimic the size, shape and surface characteristics of real viruses may be useful in assays permeability determination of biological membranes and the screening of biological membrane permeabilising drugs or compositions, avoiding the use of real pathogenic viruses. DESCRIPTION OF THE INVENTION The present inventors have developed nanoparticles that meet the criteria required, in terms of size, hydrophobicity and cationic nature, to define a good model of virus. According to the experiments carried out but the inventors, they have proved that the nanoparticle is able to pass through biological membranes like oral or nasal mucosal membranes mimicking the infection process of real viruses. The amount of nanoparticle that passes through the membrane is correlated to the permeability of the membrane. Moreover, the present inventors also proved the suitability of the nanoparticle to test the efficacy of compositions that reduce the permeability of biological membranes, being said compositions useful in the prevention of viral infections. Therefore, the nanoparticle developed by the inventors may be used in vitro as a “virus model” for determining the permeability of a membrane and for the screening of new active agents or compositions that may be used in the clinic to combat pathogenic virus infections. Thus, in one aspect, the present invention refers to a nanoparticle, hereinafter “the nanoparticle of the invention”, comprising a core composed of a metal or an inorganic oxide, and a coating comprising, preferably consisting of, the organic compound of formula (I), R1-X1-CH2-Y1-CH2-X1-R1(I) wherein, Y1is an arylene group, a C1-C5linear alkylene group or a C2-C5linear alkenylene group; X1is a heterocyclic cation group; R1is a C10-C18linear alkyl or alkenyl group, wherein the organic compound of formula (I) comprises one or several counterions to compensate the positive charges, wherein the counterion is selected from: Br-, Cl-, I-, PF6-, BF4-, CF3CO2-, CH3CO2- or SO42-; and wherein the average diameter of the nanoparticle is from 50 nm to 200 nm, as measured by dynamic light scattering (DLS). The average diameter of the nanoparticle of the invention may also be measured by transmission electronic microscopy (TEM). As used herein the term “diameter” refers to the length of the longer axis (in a plane direction) of a given nanoparticle. The diameter of the nanoparticle of the invention may be measured by DLS or TEM. Both DLS and TEM allow to measure the diameter of a nanoparticle within a plurality of nanoparticles (preferably in a dispersion), i.e. both techniques allow to measure the length of the longer axis of each nanoparticle present in a plurality of nanoparticles. As it is known, the diameter measured by DLS corresponds to the hydrodynamic diameter that is the diameter of a hypothetical hard sphere that diffuses with the same speed as the particle being measured. It is the measure of the size of a particle in a fluid, including the particle itself and any solvent or ion molecules that are associated to the particle. Therefore, the term “average diameter” refers to the arithmetic mean of the distribution of nanoparticle diameter in a plurality of nanoparticles. According to DLS and TEM method, the number of particles within predetermined diameter ranges may be counted, and an average particle diameter may be calculated therefrom. Preferably, at least 50% of the nanoparticles in the plurality have a size within the average diameter, more preferably at least 60%, more preferably at least 70%, and still more preferably at least 75% of the nanoparticles of the plurality have a diameter within the average diameter. In view of the previous paragraph, in the present invention, the average diameter refers to the “average number diameter”, used herein interchangeably. As used herein, the term “alkyl” refers to a branched or linear, and saturated hydrocarbon radical. The term “C10-C18linear alkyl” refers to a linear alkyl having 10 to 18 carbon atoms. As used herein, the term “alkenyl” refers to a branched or linear hydrocarbon radical comprising at least one double bond. The term “C10-C18linear alkenyl” refers to a linear alkenyl having 10 to 18 carbon atoms. As used herein, the term “alkylene“ refers to a divalent alkyl moiety attached to the rest of the molecule at two positions. The term “C1-C5linear alkylene” refers to a linear alkenyl having 1 to 5 carbon atoms. As used herein, the term “alkenylene” refers to a divalent alkenyl moiety attached to the rest of the molecule at two positions. The term “C2-C5linear alkenylene” refers to a linear alkenyl having 2 to 5 carbon atoms. As used herein, the term “arylene” refers to a group having at least an aromatic ring and that is attached to the rest of the molecule at two positions. Thus, the term arylene includes multicyclic groups having at least an aromatic ring and that are attached to the rest of the molecule at two positions. Examples of arylene groups include, without limitation, phenylene, benzylene, naphthylene or antracylene. In a preferred embodiment of the nanoparticle of the invention, Y1is a C1-C5linear alkylene group, a C2-C5linear alkenylene group or an arylene group selected wherein each represents the bonding point with each -CH2- in the compound of formula (I). As used herein, the term “heterocyclic cation” refers to a 3 to 15 membered ring radical, preferably 5 to 6 membered ring, with positive charge and which consists of carbon atoms and at least one nitrogen atom. Examples of heterocyclic cations include, without limitation, pyridinium, quinolinium, imidazolium, pyrazolium, triazolium, pyridazinium, pyrimidinium, pyrrolidinium, thiazolium, oxazolium, pyrazinium, piperazinium, piperidinium, pyrrolium or indolium, quinoxalinium, In a preferred embodiment of the nanoparticle of the invention, X1heterocyclic cation group is selected from , wherein each represents the bonding point with either the R1group or the -CH2- group in the compound of formula (I). In another preferred embodiment, the nanoparticle of the invention comprises a core composed of a metal or an inorganic oxide, and a coating comprising, preferably consisting of, the organic compound of formula (I), CH2-X1- R1linear alkylene group, a C2-C5linear alkenylene group or an selected from wherein each represents the bonding point with each -CH2-; X1is a heterocyclic cation group selected from , wherein each represents the bonding point with either the R1group or the -CH2- group in the compound of formula (I); R1is a C10-C18linear alkyl or alkenyl group; wherein the organic compound of formula (I) comprises one or several counterions to compensate the positive charges, wherein the counterion is selected from: Br-, Cl-, I-, PF6-, BF4-, CF3CO2-, CH3CO2- or SO42-; and wherein the average diameter of the nanoparticle is from 50 nm to 200 nm, as measured by dynamic light scattering (DLS). As mentioned before, the average diameter of the nanoparticle of the invention may also be measured by TEM. In a more preferred embodiment of the nanoparticle of the invention, the counterion is Br-. In another preferred embodiment, the nanoparticle of the invention has an average diameter from 50 nm to 150 nm, more preferably from 70 nm to 150 nm, from 90 nm to 150 nm, from 110 nm to 150 nm or from 110 nm to 140 nm, as measured by DLS .or by TEM. The core of the nanoparticle of the invention is spherical, thus when the compound of formula (I) coats the core, the whole nanoparticle of the invention is spherical. The size of the core of the nanoparticle of the invention will depend on the desired final size of the nanoparticle. Thus, in a preferred embodiment, the nanoparticle of the invention is spherical. In a spherical nanoparticle the diameter corresponds to the line between two points of the sphere that passes through its centre. Thus, in a preferred embodiment, the core of the nanoparticle of the invention has a spherical shape with an average diameter from 30 nm to 150 nm, preferably 30 to 100 nm, more preferably from 50 nm to 100 nm, as measured by DLS or TEM. In a preferred embodiment of the nanoparticle of the invention, the core is made of a metal selected from the list consisting of Au, Ag, Cu, Zn, Pt and Pd. In a more preferred embodiment of the nanoparticle of the invention, the metal of the core is Au. As used herein, the term “inorganic oxide” refers to a binary compound of oxygen with an inorganic element, wherein the inorganic element is a cation and the oxide is an anion. Inorganic elements that can conform an inorganic oxide include, without limitation, Si, Fe, Ag, Cu, Zn, Pt or Pd. In another preferred embodiment of the nanoparticle of the invention, the core is made of an inorganic oxide selected from SiO2or a metal oxide selected from Fe2O3, Fe3O4, Ag2O and ZnO. The compound of formula (I) presents an amphiphilic nature, having both hydrophilic and hydrophobic parts. The alkyl or alkenyl (R1) groups conform the hydrophobic part, while the cationic heterocycles mainly conform the hydrophilic part. Thus, the compound of formula (I) may form bilayers, wherein the hydrophobic part (R1) of the outer layer faces inward and wherein the hydrophilic part (cationic heterocycle part) is exposed. In the inner layer, the hydrophilic part interacts with the surface of the nanoparticle and the hydrophobic part faces outward. Therefore, the two hydrophobic parts interdigitate to form the bilayer. Thus, in another preferred embodiment of the nanoparticle of the invention, the compound of formula (I) forms a bilayer on the surface of the core and is non- covalently bound to the said surface. The expression “the compound of formula (I) is non-covalently bound” means that said compound is bound by non-covalent bonds. As used herein, the term “non-covalent bond” refers to the interaction between two atoms or molecules wherein no chemical reaction takes place, including electrostatic interaction, ionic interaction, hydrogen bonding, van der Waals interaction and hydrophobic interaction. In the present invention, when the coating of the nanoparticle of the invention comprises the compound of formula (I) the counterions form a layer on the surface of the core of the nanoparticle and the cationic heterocycles interact non-covalently with said counterions on the surface of the core, preferably by electrostatic interactions. Viruses such as those belonging to the family Coronaviridae, show a cationic nature (Villena, J., & Kitazawa, H. (2020). Frontiers in Physiology, 11, 699; Manfredonia, I., et al. (2020). Nucleic acids research, 48(22), 12436-12452.) The cationic nature of the surface of viruses allows them to interact with the mucosal membranes enabling the infection of organisms like human beings. The virion core diameter is about 90nm-100nm and the spike protein molecules externally oriented promote a positive zeta potential (Adamczyk, Z., Batys, P., & Barbasz, J. (2021). Current Opinion in Colloid & Interface Science, 55, 101466.). Therefore, the new nanoparticle of the invention shows the size and charge characteristics of viruses. The nanoparticle of the present invention mimics the surface charge of viruses due to the presence of the cationic heterocyclic group of the compounds of formula (I), presenting a Z potential between +40 mV and +60 mV. The term "zeta potential (Z)" refers to a measure of the surface charge of nanoparticles. The zeta potential is determined for nanoparticles suspended in an electrolyte. In such a suspension, there is a distribution of ions from the electrolyte around the nanoparticle surface. Determination of the zeta potential allows estimation of the surface charge, and this technique can be used to check the physical stability of nanoparticles (J. Jiang, G. Oberdörster, A. Elder, R. Gelein, P. Mercer, and P. Biswas, "Does nanoparticle activity depend upon size and crystal phase?," Nanotoxicology, vol.2, no.1, pp.33-42, 2008). A large positive or negative value of the zeta potential of nanoparticles indicates good physical stability of the nanoparticles, due to the electrostatic repulsion of the individual particles. A zeta potential value outside the range of -30 mV to +60 mV is generally considered to have sufficient repulsive force to achieve better colloidal physical stability. Thus, in a preferred embodiment, the nanoparticle of the invention has a zeta potential between +40 mV and +60 mV. The zeta potential is measured in aqueous solutions of the nanoparticles at neutral pH (pH=7) by electrophoresis on a Zetasizer Nano ZS (Malvern Instruments). Preferably, the measurements of DLS, TEM and electrophoresis are carried out as in the section “General methods” of the examples in the present invention. In another preferred embodiment of the nanoparticle of the invention, the compound of formula (I) is the compound of formula (Ia), (Ib), (Ic), (Id) (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik) or (II), preferably the compound of formula (Ia), represented as follows: 5 wherein “n” ranges from 8 to 16, preferably 16, and “n1” ranges from 1 to 5. The compound of formula (I) can be synthesized trough two different protocols: a) reaction of an N-alkyl heterocycle (previously formed by reaction of a N-containing heterocyle and a haloalkane) with a bis(halomethyl)arene, or b) reaction of a N- containing heterocycle with a bis(halomethyl)benzene followed by quaternization of the heterocycle by reaction with an haloalkene. In another aspect, the present invention also refers to a method for the synthesis of the nanoparticle of the invention, hereinafter “the synthesis method of the invention”, wherein said method comprises the following steps: a) Contacting a nanoparticle of a metal or an inorganic oxide with the compound of formula (I), b) Sonicating the mixture of step a), and c) Stirring the product obtained in step b). In a preferred embodiment of the synthesis method of the invention, the nanoparticle of step a) has an average diameter from 30 nm to 150 nm, preferably 30 to 100 nm, more preferably from 50 nm to 100 nm. The average size of the nanoparticle of step a) of the synthesis method of the invention is measured by DLS or TEM. In a preferred embodiment of the synthesis method of the invention, the nanoparticle of step a) is made of a metal selected from the list consisting of Au, Ag, Cu, Zn, Pt and Pd. In a preferred embodiment of the synthesis method of the invention, the nanoparticle of step a) is made of an inorganic oxide selected from Fe2O3, Fe3O4, Ag2O, ZnO and SiO2. The compound of formula (I) in the synthesis method of the inventions is the compound of formula (I) as defined in the nanoparticle of the invention or any preferred embodiment thereof. In a preferred embodiment of the synthesis method of the invention, the nanoparticle of step a) is spherical. In a preferred embodiment of the synthesis method of the invention, the nanoparticle of metal or inorganic oxide is provided in a solution at a concentration from 2 ^106particles / mL to 4 ^1011particles / mL in aqueous solution. In another preferred embodiment of the synthesis method of the invention, the compound of formula (I) is provided at a concentration from 0.05 mM to 5 mM, preferably in a solvent selected from: water or a solution of water and methanol at a 4:1 proportion. In another preferred embodiment of the synthesis method of the invention, the step b) is carried out for a time period ranging from 1 h to 3 h at room temperature (18- 25ºC). In another preferred embodiment of the synthesis method of the invention, the frequency of the sound waves on step b) is from 20 kHz to 60 kHz. In another preferred embodiment of the synthesis method of the invention, the step c) is carried out at a stirring speed from 300 rpm to 900 rpm, for a time period ranging from 6h to 24h. According to the experiments conducted by the inventors, like viruses when they penetrate the organism they infect, the nanoparticle of the invention could pass through biological membranes such as mucosal membranes, or synthetic membranes with similar characteristics to biological membranes. In addition, the present inventors were able to establish the number of nanoparticles passing through the membranes, thus determining the degree of permeability of the nanoparticles. So, in another aspect the invention refers to in vitro use of the nanoparticle of the invention for the determination of the permeability of a membrane, herein after “the permeability use of the invention”. As used herein the term “permeability of a membrane”, refers to the tendency of a membrane to allow a compound substance or particle, i.e. the nanoparticle of the invention, to pass through it. So, as it is used in the present invention, the permeability of a membrane is referred to as to the amount of nanoparticle of the invention passing through it related to time as “mg / cm2 / h”, wherein “mg” refers to the amount of nanoparticle, “cm2” refers to surface area of the membrane through which the nanoparticle passes, and “h” refers to hours. As used herein, the term "membrane" is intended to encompass the biological membranes by which a virus enters into the body of a subject, such as mucosal membranes; or synthetic membranes whose characteristics resemble to those of the biological membranes, such as pore size, by which a virus enters into the body of a subject. Synthetic membranes may be composed of any polymeric material that can form a porous film that allows the passage of aqueous solutions and nanoparticles, wherein examples of materials for synthetic membranes include, without limitation, polycarbonate, nylon or polysulfone. As used in the present invention, the term “subject” refers to an individual that may be infected by a virus, including human beings and non-human animals. Non-human animals include, without limitation, monkeys, pigs, fish, dogs, mice or rats. Thus, in another preferred embodiment of the permeability use of the invention, the membrane is a mucosal membrane. As used herein, the term “mucosal membrane” refers to the epithelial membranes which line the oral cavity, the nasal, bronchial, pulmonary, trachea and pharynx airways, the otic and ophthalmic surfaces, the urogenital system, including the prostate, the reproductive system and the gastrointestinal tract, including the colon and rectal surfaces. Reference to mucosal membrane herein is meant to include the surface membranes or cell structures of the mucosal membrane at a targeted site. In a more preferred embodiment of the permeability use of the invention, the membrane is a mucosal membrane selected from oral or nasal mucosa. In a more preferred embodiment of the permeability use of the invention, the mucosal membrane has a thickness ranging from 400 μm to 700 μm, where the thickness is measured from the surface cell layer of the membrane to the innermost cell layer included in the membrane. In another preferred embodiment of the permeability use of the invention, the membrane is a synthetic membrane having a pore size ranging from 0.050 µm to 0.450 µm. In another preferred embodiment of the permeability use of the invention, the membrane is a synthetic membrane having thickness ranging from 0.010 mm to 0.025 mm. In another preferred embodiment of the permeability use of the invention, the synthetic membrane is made of a polymer selected from polycarbonate, nylon and polysulfone. In another preferred embodiment of the permeability use of the invention, the membrane has a diameter ranging from 10 mm to 40 mm (preferably circle-shaped membrane). The present inventors tested the capacity of the nanoparticle of the invention to pass through a membrane treated with an active agent for decreasing the permeability of mucosal membranes. The aim of decreasing the permeability of a mucosal membrane is to prevent the entry of viruses into the body and therefore prevent infections caused by viruses. Therefore, the nanoparticle of the invention can be used in the study of new agents for the treatment and / or prevention of infections caused by viruses, without the need to use real viruses. Thus, in another aspect the present invention refers to the use of the nanoparticle of the invention for the in vitro screening of active agents for the treatment and / or prevention of an infection caused by a virus, hereinafter the “screening use of the invention”. As used herein, the term "active agent" refers to an agent, drug, compound or other substance, or compositions and mixtures thereof, that provide some pharmacologic, often beneficial, effect in the subject to whom it is administered. In the case of the present invention, the pharmacologic effect is the decrease of permeability of mucosal membranes of the subject. As used herein, the term “virus” refers to any infective agent that typically consists of a nucleic acid molecule with a protein coat, that is able to infect and multiply within the living cells of a subject. The pathogenic viruses usually enter the body of a subject through mucosal membranes, wherein some of these pathogenic viruses include, without limitation, respiratory viruses like coronaviruses, adenoviruses, rhinoviruses or parainfluenza viruses. In a preferred embodiment of the screening use of the invention, the virus is a respiratory virus selected from the list consisting of coronavirus, rhinovirus, influenza virus, parainfluenza virus or parvovirus. The present inventors have developed methods for the in vitro determination of the permeability of a membrane and for the in vitro screening of active agents for the prevention of an infection caused by a virus that are explained below. The terms “membrane”, “permeability of a membrane”, “active agent”, “prevention”, “virus”, as well as their particular embodiments, have been defined previously wherein that definitions are applicable in the methods described below. In another aspect, the present invention refers to a method for the in vitro determination of the permeability of a membrane, hereinafter “permeability method of the invention”, comprising the following steps: a) Contacting the membrane with the nanoparticle of the invention; and b) Quantifying the amount of nanoparticle permeated through the membrane. In the step a) of the permeability method of the invention, the nanoparticle of the invention is contacted with one surface of the membrane, wherein said nanoparticle is provided at a predetermined concentration in an aqueous solution (solution 1). Another solution not containing the nanoparticle of the invention is contacted with the other surface of the membrane (solution 2). Therefore, the solution 1 and 2 are separated by the membrane. Once the solution 1 is contacted with one of the surfaces of the membrane, the nanoparticle of the invention is permeated through the membrane reaching the solution 2 in contact with the other surface of the membrane. This process is carried out at a constant temperature similar to the body temperature of a subject that may be infected by a virus. In a preferred embodiment, the permeability method of the invention is carried out at a temperature ranging from 35 ºC to 40ºC, preferably 37 ºC. In the step b) of the permeability method of the invention, the amount of nanoparticle permeated through the membrane, i.e. the amount of nanoparticle in then solution 2, is quantified. The amount of permeated nanoparticle varies depending on the permeability of the membrane. Therefore, the amount of nanoparticle permeated through the membrane is directly related to the membrane permeability. In a preferred embodiment of the permeability method of the invention, the quantification of step b) is carried out by mass spectroscopy. In another preferred embodiment of the permeability method of the invention the membrane is a mucosal membrane, preferably oral or nasal mucosa. In a more preferred embodiment of the permeability method of the invention, the mucosal membrane has a thickness ranging from 400 μm to 700 μm, where the thickness is measured from the surface cell layer of the membrane to the innermost cell layer included in the membrane. In another preferred embodiment of the permeability method of the invention, the membrane is a synthetic membrane having a pore size ranging from 0.050 µm to 0.450 µm. In another preferred embodiment of the permeability method of the invention, the synthetic membrane has thickness ranging from 0.010 mm to 0.025 mm. In another preferred embodiment of the permeability method of the invention, the synthetic membrane is made of a polymer selected from polycarbonate, nylon and polysulfone. In another preferred embodiment of the permeability method the invention, the membrane has a diameter ranging from 10 mm to 40 mm (preferably circle-shaped membrane). In another aspect, the present invention refers to a method for the in vitro screening of active agents for the treatment and / or prevention of an infection caused by a virus, hereinafter “the screening method of the invention”, comprising the following steps: a) Contacting an active agent with a membrane; b) Contacting the membrane with a nanoparticle of the invention after the step a); and c) Quantifying the amount of nanoparticle permeated through the membrane after step b). First, in the step a) the active agent to be tested is contacted with one of the surfaces of the membrane. Then, in the step b) of the screening method of the invention, the nanoparticle of the invention is contacted with the same surface of the membrane as in the step a), wherein said nanoparticle is provided at a predetermined concentration in an aqueous solution (solution 1). Another solution not containing the nanoparticle of the invention is contacted with the other surface of the membrane (solution 2). Therefore, the solution 1 and 2 are separated by the membrane. Once the solution 1 is contacted with one of the surfaces of the membrane, the nanoparticle of the invention is permeated through the membrane reaching the solution 2 in contact with the other surface of the membrane. This process is carried out at a constant temperature similar to the body temperature of a subject that may be infected by a virus. In a preferred embodiment, the screening method of the invention is carried out at a temperature ranging from 35 ºC to 40ºC, preferably 37 ºC. In the step c) of the screening method of the invention, the amount of nanoparticle permeated through the membrane, i.e. the amount of nanoparticle in the solution 2, is quantified. The solutions used either in the permeability method or the screening method of the invention, refer to water or a buffer solution such as phosphate buffer solution a physiological pH of about 7.4. In a preferred embodiment of the screening method of the invention, steps b´) and c´) are carried out in parallel to steps b) and c). Steps b´) and c´) are carried out as the steps b) and c), but without the previous step a). A reduced amount of permeated nanoparticles in step c) compared to the amount of permeated nanoparticles in step c´) means that the active agent reduced the permeability of the membrane, and that the active agent is suitable for preventing the pass of a virus through biological membranes. In a preferred embodiment of the screening method of the invention, the quantifications of step c) and step c´) are carried out by mass spectrometry. In another preferred embodiment of the screening method of the invention the membrane is a mucosal membrane, preferably oral or nasal mucosa. In another preferred embodiment of the screening method of the invention, the membrane is a synthetic membrane having a pore size ranging from 0.050 µm to 0.450 µm. In the present invention the “term pore size”, refers to the average diameter of the pore(s) of a membrane. In another preferred embodiment of the screening method of the invention, the membrane is a synthetic membrane having thickness ranging from 0.010 mm to 0.025 mm. In another preferred embodiment of the screening method of the invention, the synthetic membrane is made of a polymer selected from polycarbonate, nylon and polysulfone. In another preferred embodiment of the screening method the invention, the membrane has a diameter ranging from 10 mm to 40 mm. Synthetic membranes suitable for performing the methods of the present invention can be commercially available, a non-limiting example of commercially available synthetic membranes is Nuclepore ®. Both the permeability method and the screening method of the invention, can be carried out using a system that comprises two connected chambers where the solutions 1 and 2 can be placed. The membrane is placed at the connection of the two chambers, thus separating the solutions. Systems known to the person skilled in the art such as, but not limited to, Franz diffusion cells, can be used to carry out the permeation method and the screening method of the invention. Thus, in a preferred embodiment, the permeability method and the screening method of the invention are carried out in a Franz diffusion cell. When using a mucosal membrane to conduct the permeability method and the screening method of the invention, said membrane refers to a sample of a mucosal membrane obtained from a subject as previously defined. The sample of a mucosal membrane can be obtained by means known by the skilled person such a dermatome, wherein the mucosal membrane is provided as a tissue portion that comprises lax connective tissue, lining epithelium, the basal and spinous layer. DESCRIPTION OF THE DRAWINGS Fig.1. a) Amphiphile compound 1 ^2Br and b) schematic representation of gold nanoparticle comprising a bilayer of the compound 1 ^2Br on the surface (AuNPS-1 ^2Br). Fig.2. UV-Vis absorption spectra for Citrate-AuNPs and 1.2Br-AuNPs. Fig.3. Zeta potential plots of (a) Citrate-AuNPs and (b) 1.2Br-AuNPs. Fig.4. Zeta size plots of (a) Citrate-AuNPs and (b) 1.2Br-AuNPs. Fig.5. TEM image of 1.2Br-AuNPs in 200 nm scale. Fig.6. TEM image of 1.2Br-AuNPs in 50 nm scale. Examples Example 1. Synthesis of the virus model For the synthesis of nanoparticles that mimic the size and surface characteristics of a virus, a procedure was followed as described below. Commercial citrate-stabilized gold nanoparticles (Citrate-AuNPs, Sigma Aldrich) were used, with a concentration ~3.8 ^109particles / mL, and 100 nm in average diameter. These nanoparticles were functionalized with bis[(3-octadecyl-1- imidazolium)methyl]benzene 1 ^2Br (Fig.1a). For the synthesis of bis[(3-octadecyl-1- imidazolium)methyl]benzene 1 ^2Br, 1H-Imidazole (0.5 g, 7.35 mmol) and potassium hydroxide (0.49 g, 8.82 mmol) were mixed in a dry acetonitrile (30 mL) under an argon atmosphere and at room temperature and stirred until fully dissolved (2 h). Then, a solution of 1-bromooctadecane (2.45 g, 7.35 mmol) in dry acetonitrile (25 mL) was added at 40 ºC. The solution was allowed to cool to room temperature and stirred for 24 h, resulting in a white solid. Afterward, the solvent was removed by evaporation, and residue was partitioned between dichloromethane (20 mL) and water (18 mL). The water phase was extracted by dichloromethane twice (2 x 40 mL) followed by washing the organic phase with water (35 mL), dried using MgSO4, and filtered. The solvent was then evaporated, resulting in the production of 1- octadecyl-1H-imidazole, a yellow oil that solidified at room temperature under vacuum (4.03 g, 87%). Afterward, 1,3-bis(bromomethyl)benzene (0.5 g, 1.89 mmol) was dissolved in dry acetonitrile (20 mL) under argon atmosphere followed by adding 1-octadecyl-1H-imidazole (1.2 g, 3.79 mmol). The reaction stirred for 24 h at 80 ºC, and was allowed to cool to room temperature. Solvent was evaporated under vacuum, resulting in a white solid (1.11 g, 96%). Herein after 1 ^2Br, through ligand exchange, by treatment with a 0.55 mM aqueous solution of the amphiphilic compound 1 ^2Br, using sonication for 2 h at a frequency of 37 kHz followed by stirring at room temperature for 12 h at 600 rpm. The overnight stirring was assayed using either an orbital shaker or a magnetic stirrer. The colloidal dispersion of 1.2 Br-AuNPs was then centrifuged for 15 minutes at 20,000 rpm and washed with Milli- Q H2O (3 × 1 mL). The pellet of 1.2 Br-AuNPs was stored in the fridge for further experiments. Example 2. Characterization The freshly prepared gold nanoparticles coated with bis[(3-octadecyl-1- imidazolium)methyl]benzene 1 ^2Br (1 ^2Br-AuNPs, Fig. 1b) were characterized by UV-vis absorption spectroscopy, dynamic light scattering (DLS), as well as transmission electron microscopy (TEM), in order to know their concentration, size distribution as well as their electrokinetic potential or surface charge, and their morphology. The data is included in Table 1. This allowed to know their gold concentration (6 ^10-12M) concentration, as well as their size in solution, which responds to a diameter of 120 nm (PDI 0.15) and its Z potential is ca.45mV, which indicates their cationic nature and their high stability. Table 1. UV-vis absorption spectroscopy data, size, and zeta potential values for 1 ^2Br-AuNPs. 1 Wavelength corresponding to the absorption of the Surface plasmon resonance (SPR) band.2Determined by UV-vis absorption spectroscopy.3Measured by DLS. 4Standard deviation.5Polydispersity index. Fig. 2 exhibits UV-Vis absorption spectra for Citrate-AuNPs and 1.2Br-AuNPs. The maximum absorbance peak for both functionalized gold nanoparticles appears about 570 nm which shows that there is not any change in the gold core after ligand exchange process. The molar concentration of gold is also calculated 6.4 × 10-12M. Fig.3 and Fig. 4 also show the zeta potential and dynamic light scattering (DLS) plots, respectively, for Citrate-AuNPs and 1.2Br-AuNPs. As it can be seen, the zeta potential average changed from a negative value for Citrate-AuNPs to a positive value for 1.2Br-AuNPs. Moreover, the zeta potential average for 1.2Br-AuNPs is + 42 mV which was more positive than the threshold (±25 mV) for the colloidal stability of nanoparticles. According to DLS results (Table 1 and Figure 4), there is 8 nm increment in the hydrodynamic diameter gold nanoparticles indicating the formation of a bilayer of cationic 1.2Br compound around gold nanoparticles (Fig 1b). Fig.5 and Fig.6 also exhibit the negative staining TEM images of 1.2Br-AuNPs in two different scales of 200 nm and 50 nm, respectively. As it can be seen in Fig.6, the layer around gold nanoparticle corresponds to a bilayer of cationic 1.2Br compound, which is shown by the negative staining method. Example 3. Virus model permeation The feasibility of the gold nanoparticles previously described and synthesized, according to examples 1 and 2, as a virus model of the was studied, both with artificial membrane and with biological membranes. Synthetic membrane (Nuclepore®), buccal mucosa and sublingual mucosa were used as permeation membrane. The biological membranes were surplus from surgical practice from the Faculty of Medicine, University of Barcelona, Campus Bellvitge. The manipulation of the mucosa is carried out following the basic safety regulations for protection against possible exposure to pathogenic viral agents. The material used (scissors, scalpel, etc.) is sterilized in the steribox at 150 ºC for 20 minutes while the remains of biological material are removed in special containers that are at low temperatures. The oral mucosa is cut into strips of about 3-4 cm and 500 µm thick using an electric dermatome. This thickness allows the entire mucosal barrier to be represented and therefore to study drug passage through the loose connective tissue, lining epithelium, basal layer, and spinous layer. A Franz cell was used in order to perform the permeability experiments, wherein the Fran cell comprised a recipient and a donor compartment. The mucosa is placed with the underside in contact with the fluid of the receiving compartment, therefore the compartments of the Franz cell were separated by the membrane. The recipient compartment is filled with water and 100 mg of Formulation 3 (F3) protectant formulation is placed in the donor compartment. F3 was a composition that consisted of the following components, whose amount is expressed in weight % respect to the total weight of the composition: - 10 % of stringy Vaseline; and - 90 % of isopropyl myristate. Next, 0.4 mL microliters of the suspension of the virus model were sown. Said suspension has a concentration of 0.595 µg / mL. Subsequently, the upper part of the cell is covered with Parafilm®. The temperature is maintained inside the cell at 37°C ± 0.5. The sample is taken manually through the side tube of the Franz cell, at predetermined intervals of every hour for 6 hours. The volume that has been extracted is immediately added with the replacement medium. After completion of the permeation assay, the membrane was removed from the Franz cells and the excess formulation was removed from the membrane surface. This was cleaned with gauze soaked in 0.05% sodium lauryl sulfate solution and washed 3 times with distilled water. The skin pieces were placed in glass vials and the virus content was extracted with water. The supernatants were pipetted and analyzed, obtaining the amount of virus retained in the mucosa. The extractions were placed in chromatography vials, until their analysis by plasma mass spectrometry. Their determination was carried out according to the following methodology: in Teflon capsules (cleaned with Aqua Regia), they were weighed and tared and later, the sample was weighed and 0.5 ml of Agua Regia was added. Left overnight at 90 °C. After cooling, 10 ml Thiourea (200ppm 1% HCl) was added. Finally, it was covered, homogenized and weighed, and passed to glass tubes, washing the tubes with the attack solution. The analysis was performed by inductively coupled plasma mass spectrometry with the Agilent Technologies Inc. - 7500ce ICP-MS equipment. Table 2 shows the virus retained (amount present on the membrane after completion of the assay) and permeated (amount present in the medium of the acceptor compartment of the cell) after its deposition on the different membranes. The results of the modified membranes correspond to the values of the virus deposited on membranes previously treated with the F3 Base lipophilic MI formulation. Among all the formulations tested, this was the one that gave rise to the lowest TWEL values. For this reason, it is selected as the first candidate to be applied on mucous membranes and study its protective character against the virus model. Table 2. Amounts of retained and permeated virus on membranes and modified membranes with an waterproofing formulation, expressed as gold µg / mL In both results, retention and permeation, the barrier action of the waterproofing formulation is observed for all membranes. This is especially relevant in the permeation of the virus, with much lower values for the waterproofed artificial and oral membranes. Which means great protection against the passing of the virus model. On the other hand, a great similarity is observed between the artificial membrane and oral mucosa for the permeated virus model. This similarity is maintained before and after the treatment of the membranes with the waterproofing formulation. Based on this, the artificial membrane could be used instead of the biological one for permeation tests. Materials 100 nm diameter citrate-capped gold nanoparticles (Citrate-AuNPs) were purchased from Sigma Aldrich. The water used in the experiments was HPLC grade, produced by a Milli-Q plus system from Millipore (Milli-Q water). General methods UV-vis absorption spectroscopy was performed using a UV-1800 Shimadzu UV spectrophotometer. Dynamic light scattering (DLS) was measured in water at ambient temperature using a Zeta Sizer Nano series (Malvern Co.). Zeta potential was also measured using a Zeta Sizer Nano series (Malvern Co.) in 6 mM NaCl solution at ambient temperature. Transmission electron microscopy (FEI Tecnai G2 F20200 kV) was utilized for acquiring TEM images. Carbon film-supported copper grids using a negative staining method (2% uranyl acetate) were employed for the TEM sample preparation. Nanoparticle centrifugation was performed using Eppendorf AG 22331 Hamburg.

Claims

CLAIMS 1. A nanoparticle comprising a core composed of a metal or an inorganic oxide, and a coating comprising the organic compound of formula (I), Y1-CH2-X1-R1wherein, Y1is an arylene group, a C1-C5linear alkylene group or a C2-C5linear alkenylene group; X1is a heterocyclic cation group; R1is a C10-C18linear alkyl or alkenyl group, wherein the organic compound of formula (I) comprises one or several counterions to compensate the positive charges, wherein the counterion is selected from: Br-, Cl-, I-, PF6-, BF4-, CF3CO2-, CH3CO2- or SO42-; and wherein the average diameter of the nanoparticle is from 50 nm to 200 nm, as measured by dynamic light scattering (DLS).

2. Nanoparticle according to claim 1 wherein:CH2-;X1is a heterocyclic cation group selected from and , wherein each represents the bonding point with either the R1group or the -CH2- group in the compound of formula (I); R1is a C10-C18linear alkyl or alkenyl group; wherein the organic compound of formula (I) comprises one or several counterions to compensate the positive charges, wherein the counterion is selected from: Br-, Cl-, I-, PF6-, BF4-, CF3CO2-, CH3CO2- or SO42-; and wherein the average diameter of the nanoparticle is from 50 nm to 200 nm, as measured by dynamic light scattering (DLS).

3. Nanoparticle according to any one of claims 1 or 2, wherein the average diameter of the core is from 30 nm to 150 nm.

4. Nanoparticle according to any one of claims 1 to 3, wherein the metal of the core is selected from the list consisting of Au, Ag, Cu, Zn, Pt and Pd; or the inorganic oxide of the core is selected form Fe2O3, Fe3O4, ZnO and SiO2.

5. Nanoparticle according to any one of claims 1 to 4, wherein the core consists of Au.

6. Nanoparticle according to any one of claims 1 to 5, wherein the counterion is Br-.

7. Nanoparticle according to any one of claims 1 to 6, wherein the compound of formula (I) forms a bilayer on the surface of the core and is non-covalently bound to said surface.

8. Nanoparticle according to any one of claims 1 to 7, wherein the compound of formula (I) is the compound of formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik) or (II) represented as follows:wherein “n” ranges from 8 to 16 and “n1” ranges from 1 to 5.

9. Use of the nanoparticle according to any one of claims 1 to 8 for the in vitro determination of the permeability of a membrane.

10. Use for the in vitro determination of the permeability of a membrane according to claim 9, wherein membrane is a mucosal membrane or a synthetic membrane, preferably wherein the mucosal membrane is selected from oral mucosa and nasal mucosa.

11. Use of the nanoparticle according to any one of claims 1 to 8 for the in vitro screening of active agents for the prevention of an infection caused by a virus.

12. Method for the in vitro determination of the permeability of a membrane comprising the following steps: a) Contacting the membrane with a nanoparticle according to any one of claims 1 to 8; and b) Quantifying the amount of nanoparticle permeated by the membrane.

13. A method for the in vitro screening of active agents for the prevention of an infection caused by a virus, comprising the following steps: a) Contacting an active agent with a membrane; b) Contacting the membrane with a nanoparticle according to any one of claims 1 to 8, after the step a); and c) Quantifying the amount of nanoparticle permeated by the membrane after step b).

14. Method according to any one of claims 12 or 13, wherein membrane is a mucosal membrane or a synthetic membrane, preferably wherein the mucosal membrane is selected from oral mucosa and nasal mucosa.

15. A method for obtaining a nanoparticle according to any one of claims 1 to 8, comprising the following steps: a) Contacting a nanoparticle of a metal or an inorganic oxide with the compound of formula (I), b) Sonicating the mixture of step a), andc) Stirring the product obtained in step b).

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