Synthetic viral nanostructures and uses thereof

Abstract

The present invention relates to a nanostructure comprising a magnetic core coated by one or more cellular components, wherein said magnetic core comprises magnetic nanoparticles. The invention further relates to a method for producing said nanostructure, and to use of said nanostructure as a medicine, as a vaccine, or for the in vitro analysis of compounds capable of inhibiting viruses, for in vitro antibody validation or as an in vitro system for intracellular biopsy.

Description

[0001] DESCRIPTION

[0002] Synthetic viral nanostructures and their uses

[0003] The present invention pertains to the biomedical field, particularly immunology, vaccine development, and analysis of viral inhibitors. The present invention relates to synthetic viral nanostructures composed of a magnetic core coated with a cell membrane containing viral ligand proteins.

[0004] BACKGROUND OF THE INVENTION

[0005] Experimentation with pathogenic viruses is necessary to understand the potential risks posed by viruses, for the development of inhibitory drugs and vaccines, and to understand how they spread and cause disease (Yeh KB., et al., Significance of High-Containment Biological Laboratories Performing Work During the COVID-19 Pandemic: Biosafety Level-3 and -4 Labs. Front Bioeng Biotechnol. Frontiers Media SA; 2021;9:731).

[0006] Although these studies are necessary to improve our understanding of viruses and develop effective treatments and vaccines, it is important that they be conducted under strict safety measures, due to the risks involved in working with highly infectious and potentially lethal microorganisms. These studies are conducted in Biosafety Level III (P3) laboratories, also known as BSL-3 (biosafety level 3 laboratories), which are specialized laboratories with a high level of safety. They allow for rapid research to characterize human and animal pathogens, assist in disease surveillance, and conduct initial preclinical research for the development of diagnostics, therapies, and vaccines.

[0007] However, these laboratories present numerous technical and financial challenges, as access to them requires consistent and rigorous external certifications, training of specialized personnel, and exhaustive controls and constant monitoring (air flow, pressure, water flow, temperature, and power) (X / a H, Yuan Z. High-containment facilities and the role they play in global health security. J Biosaf Biosecurity. Elsevier; 2022;4:1-4). Furthermore, the design, construction, and commissioning of these laboratories are costly and require a large investment. Therefore, they are rare in research institutes, universities, or clinics, thus limiting R&D studies against these pathogens.This is a problem in pandemics like the one suffered by SARS-CoV-2, where intensive study and understanding of the virus's pathological mechanisms is required in order to develop efficient protection systems.

[0008] In addition to the problems we've discussed regarding the manipulation of viruses for study, it is first necessary to obtain a sample of the virus and its different variants, usually from clinical samples. This significantly limits access to this material for generating inhibitory drugs or vaccines. Another handicap is that working with viruses requires obtaining specimens from clinical samples, and if there are different genetic variants, taking samples is not always possible (due to availability issues) or safe.

[0009] On the other hand, to generate protective vaccines against enveloped human pathogens, it is essential to understand how antibodies interact with viral envelope proteins, particularly fusion proteins, and how to neutralize these viruses with antibodies. Creating recombinant protein-based vaccines involves producing a specific viral protein in a cellular expression system, which is then purified and used as an antigen in the vaccine. Specifically, recombinant proteins such as the SARS-CoV-2 Spike or "S" protein have numerous disadvantages. Recombinant protein production is generally considered a safe and efficient method for obtaining recombinant proteins on a large scale, but some drawbacks must be taken into account.First, the process of producing and purifying recombinant proteins composed of polypeptide varietals can be expensive, time-consuming, and requires specific equipment and technical training, as they cannot be produced in bacteria due to poor folding and lack of glycosylation. Specifically, the Spike or "S" protein must be produced in human cells, and its production is slow, with a high risk of bacterial and fungal contamination, affecting the quality of the final product.

[0010] Viruses such as coronavirus (enveloped viruses) consist of a capsule containing genetic material surrounded by a bilaminated lipid membrane with ligand proteins (e.g., in SarCov2, the Spike or "S" protein) incorporated into the virus when the virus buds outside the cells it has infected (host cells). Most pathogenic viruses are covered by these membranes, which mediate adhesion to the surface of their target cells and allow the fusion of the viral envelope with the lipid bilayer of the membrane of the cells they are infecting. Each virus has different ligand proteins, but their purpose is the same: to direct the virion to receptors on target cells for replication.

[0011] In short, the ideal would be to have a synthetic system, a kind of viral avatar, incapable of replication, but externally identical to the virus in size and surface properties, so that the target cells of these microorganisms would recognize it in the same way as the virus itself.

[0012] In view of all the above, it would be necessary to develop synthetic and biosafety nanosystems for experimentation with the virus and its diseases, as well as for the development of vaccines.

[0013] DESCRIPTION OF THE INVENTION

[0014] The present invention relates to magnetic nanostructures, preferably spherical, oval, or square, with dimensions similar to a virus (50 to 500 nm in diameter), which can be fluorescent, and are made of silica or polystyrene. Thanks to their magnetic properties, their entry into and exit from cells can be facilitated by using magnetic force. Additionally, the nanostructures can be visualized using electron microscopy techniques (Figure 1) and coated with viral proteins that bind enveloped viruses, thus forming a system incapable of replication but extremely identical to the virus, thus obtaining a "viral avatar."

[0015] To obtain the magnetic nanostructures described above, magnetic and fluorescent nanoparticles are added to a cell culture, preferably recombinant cells that express viral proteins binding enveloped viruses on their surfaces, and are forced into the cell interior using a magnetic field perpendicular to the base of the cell growth plate. The nanoparticles are subsequently extracted from the cell interior by shifting or reapplying the magnetic field to the top of the culture, causing said nanoparticles to emerge by forced budding, in an effect similar to viral budding, thus obtaining the aforementioned magnetic nanostructures comprising a cellular component on their surface. These nanostructures, once collected, for example, using a magnetic field, can be used in cell interaction studies, viral inhibitor assays, or immunization assays (Figure 2).In an example of the present invention, these nanostructures are surrounded by a crown of Spike protein identical to that of the coronavirus under study (Figure 3).

[0016] This safe, scalable, and easy-to-handle nanobiotechnology-based system provides the same size and envelope characteristics as viruses, but lacks infectious capacity (no genetic material), so they can be called "viral avatars." This allows these nanostructures to be used both in immunization and in research into viral inhibitors or antibody validation, avoiding all the biosafety issues that would be necessary when using viral particles. Therefore, the nanostructures of the present invention can be used in: (i) immunization; (ii) studies of compounds for inhibiting the same penetration mechanisms of viruses; (iii) studies for the selection of antibodies against different viruses; and (iv) development of immunodetection kits, among other applications.

[0017] The versatility of the system is enormous, as it allows for the production of nanostructures structurally similar to viruses that comprise any cellular component on their surface, as well as for the expression of any viral membrane ligand protein by transfecting the human cells in the culture, which allows the system to be applied to any enveloped virus.

[0018] Nanostructure of the invention

[0019] In view of the foregoing, in a first aspect, the present invention relates to nanostructures, hereinafter “the nanostructures of the invention”, comprising a nanostructure comprising a magnetic core coated by one or more cellular components, wherein said magnetic core comprises magnetic nanoparticles.

[0020] The term "nanostructures," as used herein, refers to structures, systems, or complexes that have dimensions in the range of 1 to 1000 nm. Preferably, the nanostructures of the invention are characterized by having an average diameter of between 5 and 500 nm (ends included). This size allows the nanostructures to have dimensions similar to viruses and to facilitate cell entry and exit. In a preferred embodiment, the nanostructures of the invention have a size between 50 nm and 500 nm (ends included), more preferably between 100 nm and 200 nm (ends included).

[0021] In the present invention, the term “magnetic core” refers to the central compartment of the nanostructure of the invention, which comprises a support medium and magnetic nanoparticles.

[0022] The nanostructures of the invention comprise magnetic nanoparticles. The term "magnetic nanoparticles," as used herein, refers to nanoparticles that can be manipulated by external magnetic fields; that is, their behavior and position can be controlled using a magnetic field generated from outside the nanoparticle, and they have the ability to respond to external magnetic influences.

[0023] In a preferred embodiment of the nanostructures of the invention, the magnetic nanoparticles are iron oxide nanoparticles.

[0024] In another preferred embodiment of the nanostructures of the invention, the support of the magnetic core comprises at least one polystyrene sphere (hereinafter, “polystyrene nanostructure of the invention”, see Figure 5) or at least one silica sphere (hereinafter, “silica nanostructure of the invention”, see Figure 6) in combination with the magnetic nanoparticles, more preferably, iron oxide magnetic nanoparticles.

[0025] Polystyrene nanostructure of the invention

[0026] In another preferred embodiment of the nanostructure of the invention, the magnetic core comprises at least one polystyrene sphere as a support medium for the magnetic nanoparticles. The polystyrene nanostructure of the invention is shown in Figure 5.

[0027] The term "polystyrene sphere," as used herein, refers to spherical nanoparticles of the polymer polystyrene, composed of repeating units of styrene, an aromatic hydrocarbon monomer. Polystyrene is highly biocompatible, making it a key component of medical and biomedical applications, such as medical devices, implants, and other products that will come into direct or indirect contact with tissues and biological fluids.

[0028] In a particular embodiment of the polystyrene nanostructure of the invention, the polystyrene sphere is surrounded by at least 2, 3 or 4 layers of polyelectrolytes.

[0029] The term “polyelectrolyte” as used herein refers to any polymer having electrolyte groups, i.e. polycations and polyanions. These groups dissociate in aqueous solutions (water), leaving them as charged polymers. Examples of positively charged polyelectrolytes include, but are not limited to, polyethyleneimine, poly(allylamine hydrochloride), poly(diallyldimethylammonium chloride) or PPDA, poly(acrylic acid). Examples of negatively charged polyelectrolytes include, but are not limited to, polystyrene sulfonate, polyacrylamide, polyacrylic acid, polymethacrylic acid, poly(sodium 4-styrene sulfate or PSS), poly-(styrenesulfonate), poly(sodium sulfate), poly(sodium acrylate).

[0030] Any polyelectrolyte can be used in the present invention. However, in another even more particular embodiment of the polystyrene nanostructure of the invention, the polyelectrolyte layer comprises poly(sodium 4-styrene sulfate) (PSS) and / or poly(diallyldimethylammonium chloride) (PPDA).

[0031] The terms “poly(sodium 4-styrene sulfate)”, “poly(sodium styrene sulfonate)”, “sodium polystyrene sulfonate” or “PSS”, as used herein, refer to a substituted polystyrene polymer in which a SO sulfonate group is bonded to each n4 carbon atom of the phenyl group. It has the appearance of a solid or white powder, very soluble in water. It is obtained by polymerization or copolymerization of styrene sulfonate, or by sulfonation of already polymerized polystyrene.

[0032] The terms “poly(diallyldimethylammonium chloride)” or “PPDA” as used herein refer to a cationic polyelectrolyte prepared by the radical polymerization of diallyldimethylammonium chloride in the presence of a catalyst in the form of organic peroxide.

[0033] PPDA and PSS can be used in combination in the polystyrene nanostructure of the invention to form a layer-by-layer adsorbed film of negatively and positively charged polymers. Thus, in another preferred embodiment of the polystyrene nanostructure of the invention, the polyelectrolyte layers alternate between a layer of PPDA and a layer of PSS.

[0034] Additionally, the PPDA of the polystyrene nanostructure of the invention may be bonded to a fluorochrome so that the polystyrene nanostructure of the invention can be visualized or identified by fluorescence. Thus, in a particular embodiment of the polystyrene nanostructure of the invention, the nanostructure comprises a bilayer of PSS and PPDA, where the PPDA is bonded to a fluorochrome. This bilayer of PSS and PDA-Fluorochrome may be additional to other polyelectrolyte layers present in the polystyrene nanostructure of the invention.

[0035] In the present invention, a fluorophore is understood to mean a photoreactive chemical compound that absorbs light energy of a certain wavelength and emits that light at a broader wavelength. In general terms, fluorochromes are functional groups that are attached to another molecule, which allows the visualization of said molecules by fluorescence. Examples of fluorochromes include, but are not limited to, fluorescein, rhodamine, cyanine 5 (Cy 5), Tetramethyl Rhodamine Isothiocyanate (TRITC), Alexa Fluor series, Pacific Blue and any of them can be used in the present invention. However, in another even more particular embodiment, the fluorochrome is fluorescein isothiocyanate (FITC).

[0036] In another preferred embodiment of the polystyrene nanostructure of the invention, the electrolyte layers are surrounded by the magnetic nanoparticles.

[0037] Briefly, in a preferred embodiment of the nanostructure of the invention, the nanostructure comprises a magnetic core coated by a cellular component, where

[0038] (i) the magnetic core comprises

[0039] (a) magnetic nanoparticles, preferably iron oxide nanoparticles,

[0040] (b) at least one polystyrene sphere, and

[0041] © at least 2, 3, or 4 polyelectrolyte layers; preferably, the polyelectrolyte layer comprises PSS and / or PPDA, where the polystyrene sphere is surrounded by the polyelectrolyte layers which, in turn, are surrounded by the magnetic nanoparticles; and

[0042] (i) the cellular component. Silica nanostructure of the invention

[0043] In another preferred embodiment of the nanostructure of the invention, the magnetic core support comprises at least one silica sphere. The silica nanostructure of the invention is shown in Figure 6.

[0044] The term “silica sphere,” as used herein, refers to spherical silica nanoparticles (e.g., silicon dioxide, SiO2) with a size in the nanometric range. Silicon dioxide is a chemical compound composed of one silicon atom and two oxygen atoms, and is one of the most common components of the Earth's crust. Silicon is the element closest to carbon, and in fact, many carbon compounds have homologous equivalents to silicon. Colloidal silica is highly biocompatible and is currently used as an additive in food production and in the cosmetics and pharmaceutical industries.

[0045] The silica nanostructure of the invention comprises a silica sphere, in which the magnetic nanoparticles are located inside the silica sphere. Thus, in a particular embodiment of the silica nanostructure of the invention, the silica sphere comprises the magnetic nanoparticles inside, preferably at least two magnetic nanoparticles.

[0046] Likewise, as understood by those skilled in the art, the silica nanostructure of the invention can be fluorescent if it comprises a fluorophore, which allows for easy identification. Thus, in another preferred embodiment of the silica nanostructure of the invention, the silica sphere comprises a fluorophore, more preferably, the fluorophore is FITC. The term "fluorophore" and examples of fluorophores that can be used in the context of the present invention have been described in previous paragraphs.

[0047] Briefly, in a preferred embodiment of the nanostructure of the invention, the nanostructure comprises a magnetic core coated by a cellular component, where

[0048] (i) the magnetic core comprises

[0049] (a) magnetic nanoparticles, preferably iron oxide nanoparticles, and

[0050] (b) at least one silica sphere, where the silica sphere comprises the magnetic nanoparticles inside; and

[0051] (i) the cellular component.

[0052] As can be seen, the nanostructure of the invention, whether it comprises polystyrene spheres or silica spheres, comprises a cellular component.

[0053] In the present invention, "cellular component" refers to any compound, structure, or part of a cell. Examples of cellular components include, but are not limited to, nucleic acids, proteins, sugars, cell membranes, organelles of the cell cytoplasm, etc. Thus, in a preferred embodiment of the nanostructure of the invention, the cellular component is a cell membrane, a nucleic acid, or a component of the cell cytoplasm, such as nucleic acids, cytoplasmic proteins, cell organelles, etc.

[0054] In another preferred embodiment of the nanostructure of the invention, alone or in combination with all or each of the other preferred embodiments mentioned above, the cellular component is a cell membrane, where the cell membrane comprises at least one ligand protein of an enveloped virus.

[0055] In the present invention, the term "cell membrane," also known as "cytoplasmic membrane," refers to the semipermeable lipid bilayer that separates the interior of the cell from the surrounding environment. As is well known, the cell membrane regulates the transport of substances into and out of the cell. In the context of the present invention, the cell membrane of the nanostructure of the invention comprises at least one protein ligand of an enveloped virus.

[0056] In the present invention, “enveloped virus” refers to a virus that comprises a lipid envelope, or bilayer (also called “lipid envelope”, “lipid membrane”, “viral membrane” or “viral envelope”) surrounding the viral capsid, whether animal or plant viruses or bacteriophages. Said lipid envelope comprises glycoproteins or glycoproteins, i.e., ligand proteins, which are encoded in the viral genome and which help the virus enter the host cell by identifying and binding to receptors on the membrane of said host cell. Examples of enveloped viruses include, but are not limited to, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); influenza virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human immunodeficiency virus (HIV), dengue virus (DENV), or rabies virus.

[0057] Thus, in a preferred embodiment of the nanostructures of the invention, the enveloped virus is selected from the list consisting of: severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2); influenza virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human immunodeficiency virus (HIV), dengue virus (DENV), and rabies virus.

[0058] Examples of ligand proteins that may be derived from enveloped viruses include, but are not limited to, the S protein (spike protein, SARS-CoV-2 ligand protein), the hemagglutinin (H) and neuraminidase (N) proteins (in the influenza virus), surface glycoproteins C, B and D (in the herpes virus), the gp120 glycoprotein (a crucial protein in the infection process), the E protein (in the Dengue virus), or the G glycoprotein (rabies virus). However, in a preferred embodiment of the nanostructure of the invention, the ligand protein of an enveloped virus is the S protein (spike protein, SARS-CoV-2 ligand protein).

[0059] The cellular component may also be a component of the cell cytoplasm, such as nucleic acids, cell organelles, or cytoplasmic proteins.

[0060] The cellular component of the nanostructure of the invention may also be a nucleic acid, which may be either RNA or DNA, cellular organelles, cytoplasmic proteins, etc. Thus, in a preferred embodiment of the nanoparticle of the invention, alone or in combination with all or each of the previous preferred embodiments, the nucleic acid is DNA or RNA,

[0061] Composition of the invention

[0062] As understood by one skilled in the art, the nanostructures of the invention may be comprised in a composition.

[0063] Therefore, another aspect of the invention is a composition comprising the nanostructures of the invention, hereinafter the “composition of the invention”.

[0064] The nanostructures of the invention have been described hereinbefore, and they apply equally to this aspect of the invention, as well as to all its preferred embodiments, both alone and in combination with each other. In particular, the composition of the invention may be a pharmaceutical composition for use as a medicament, for example, in immunization. Thus, in a preferred embodiment, the composition of the invention is a pharmaceutical composition.

[0065] The term "pharmaceutical composition" refers to a set, mixture, or combination of components or substances comprising the nanostructures of the invention at any concentration. The pharmaceutical composition may be for human use. The term "pharmaceutical composition for human use" refers to a composition, substance, or combination of substances with properties suitable for use as a medicine, particularly as a medicine, or that can be used in humans or administered to humans for the purpose of restoring, correcting, or modifying physiological functions by exerting a pharmacological, immunological, or metabolic action, or for establishing a medical diagnosis.

[0066] In another preferred embodiment, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier and / or excipient.

[0067] The term "vehicle" or "carrier" refers to a substance, preferably an inert substance, that facilitates the incorporation of other compounds, allows for improved dosage and administration, or improves the consistency and shape of the pharmaceutical composition for use as a medicine. Therefore, a vehicle is a substance used in a medicine to dilute any of the components of the pharmaceutical composition to a specific volume or weight; or even without diluting said components, it is capable of allowing for improved dosage and administration or giving consistency and shape to the medicine. When the presentation form is liquid, the pharmaceutically acceptable vehicle is the diluent.

[0068] The term "excipient" refers to a substance that aids in the absorption of any of the components of the pharmaceutical composition, stabilizes said components, modifies their organoleptic properties, or determines the physicochemical properties and bioavailability of the pharmaceutical composition. Thus, excipients may serve the function of holding the components together, such as starches, sugars, or cellulose; sweetening; coloring; protecting the medication, such as isolating it from air and / or moisture; filling a tablet, capsule, pill, or any other form of presentation, such as dibasic calcium phosphate; and disintegrating to facilitate the dissolution of the components; without excluding other types of excipients not mentioned in this paragraph.

[0069] Furthermore, as understood by those skilled in the art, the excipient and / or vehicle must be pharmaceutically acceptable. The term "pharmaceutically acceptable" means that the vehicle or excipient must allow the compounds in the pharmaceutical composition, particularly the nanostructure of the invention, to function properly; that is, it must be compatible with said components, so that it does not cause harm to the organisms to which it is administered.

[0070] The pharmaceutical composition can be presented in any clinically permissible form of administration and in a therapeutically effective amount. For example, it can be in a form adapted for oral, sublingual, nasal, intrathecal, bronchial, lymphatic, rectal, transdermal, intravenous, intraperitoneal, orogastric, intracolonic, inhaled, or parenteral administration.

[0071] Uses of the nanostructure and composition of the invention

[0072] Medical uses of the invention

[0073] As explained at the beginning of this description, the nanostructures of the invention can be used as a medicine. Therefore, another aspect of the invention relates to the nanostructures or composition of the invention for use as a medicine.

[0074] The term "medicament," as used herein, refers to any substance used for the prevention, alleviation, treatment, or cure of disease in a subject, or that can be administered to the subject, for the purpose of restoring, correcting, or modifying its physiological functions by exerting a pharmacological, immunological, or metabolic action. For the purposes of the present invention, the terms "medicament" and "pharmaceutical composition" are used synonymously.

[0075] Likewise, another aspect of the present invention relates to the use of the nanostructures of the invention, or the composition of the invention, for the manufacture of a medicine.

[0076] In another aspect, the present invention relates to the nanostructures or composition of the invention, for use as a vaccine.

[0077] The term "vaccine" as used herein refers to a nanostructure or composition that, when administered to a subject, induces a cellular and / or humoral immune response.

[0078] In a particular embodiment, the vaccine is against SARS-CoV-2.

[0079] Non-medical uses of the invention

[0080] As described herein, the advantages of the nanostructures of the invention include, in addition to their medical use as a vaccine, their use in the study, or screening, of compounds for the inhibition of virus penetration mechanisms, of antibodies against different viruses, or their use as an intracellular "biopsy" system by extracting genetic material.

[0081] In the present invention, "intracellular biopsy" refers to the extraction of genetic material from the cell nucleus or some component of the cell cytoplasm. Examples of components of the cell cytoplasm have been defined and explained in previous paragraphs of this description.

[0082] Thus, another aspect of the present invention relates to the use of the nanostructures of the invention for the in vitro analysis of compounds capable of inhibiting viruses, for the in vitro validation of antibodies or as an in vitro intracellular biopsy system.

[0083] In the present invention, the term "in vitro" refers to experiments carried out in a controlled environment outside the living organism, such as in cell cultures. The term "in vitro analysis of compounds capable of inhibiting viruses" refers to the study in cell cultures of compounds that interfere with the binding of the virus to recipient human cells, which may lead to the development of effective medications to treat the infection.

[0084] In the present invention, the term “antibody validation” refers to the study of different antibodies to detect the body’s immune response to infection by this virus, ensuring the specificity, sensitivity, and accuracy of antibody tests.

[0085] Method for obtaining the nanostructure of the invention.

[0086] In another aspect, the present invention relates to a method for obtaining the nanostructure of the invention, hereinafter “method of the invention”, which comprises the following steps:

[0087] (a) contacting magnetic nanoparticles with a support medium to obtain a magnetic core,

[0088] (b) contacting the magnetic core obtained in (a) with a culture medium comprising a cell line;

[0089] (c) applying a magnetic field to the cells in the culture medium, thus forcing the magnetic nucleus into the cell;

[0090] (d) remove those magnetic nuclei that after step (c) have not entered the cells, and

[0091] (e) applying a magnetic field to the culture medium again to extract the magnetic nuclei from the cell interior, obtaining after the extraction a culture medium comprising the nanostructure of the invention.

[0092] Optionally, the method for obtaining the nanostructure of the invention may comprise a step (f) comprising isolating the nanostructure of the invention from the culture medium. Preferably, said isolation is carried out by centrifuging the culture medium obtained in (e), washing it with saline solution, and filtering it.

[0093] As explained throughout this description, the nanostructure of the invention comprises a magnetic core that can be based on polystyrene spheres or silica spheres that act as a support medium for the magnetic nanoparticles. Thus, depending on the nature of the magnetic core to be obtained, one method or another is carried out, which will be explained below.

[0094] Method for obtaining the polystyrene nanostructure of the invention In the event that the nanostructure is based on polystyrene spheres, in a particular embodiment the method of the invention comprises, prior to step (a), mixing at least one polystyrene sphere with an aqueous polyelectrolyte solution by stirring by sonication with low intensity ultrasonic waves for a time between 0.5 and 4 hours, thus obtaining a complex comprising the polystyrene sphere surrounded by a layer of polyelectrolytes. This step can be repeated as many times as necessary to give rise to multiple layers of polyelectrolytes. Thus, in a preferred embodiment of the method of the invention, the step comprising mixing at least one polystyrene sphere with an aqueous polyelectrolyte solution is carried out at least 2, 3, 4 or 5 times.

[0095] The polystyrene sphere is mixed with an aqueous polyelectrolyte solution by sonication stirring with low intensity ultrasonic waves, preferably between 20-95 PM; for a time between 0.5 and 4 hours, obtaining a complex comprising the polystyrene sphere surrounded by a layer of polyelectrolytes.

[0096] The term “polyelectrolyte”, as well as examples of polyelectrolytes, has been previously defined in the present description, and said definition is applicable to the method of the invention, as well as its preferred embodiments.

[0097] Thus, in a particular embodiment of the method of the invention, the aqueous polyelectrolyte solution comprises sodium poly-4-styrene sulfate (PSS) and / or poly(diallyldimethylene ammonium chloride) (PPDA). Similarly, the terms PSS and PPDA, and their preferred embodiments, have been defined in previous paragraphs.

[0098] The concentration of polyelectrolytes in the aqueous solution can vary greatly. However, in a preferred embodiment of the method of the invention, alone or in combination with the other preferred embodiments, the concentration of polyelectrolytes in the aqueous solution is between 0.5 and 3 mg / mL in 0.1 and 1 M NaCl. In another even more particular embodiment of the method of the invention, the concentration of polyelectrolytes in the aqueous solution is 2 mg / mL in 0.5 M NaCl.

[0099] Like the concentration of polylectrolytes, the concentration of magnetic nanoparticles can vary greatly and will depend on the amount of magnetic force that the person skilled in the art desires for the nanostructure of the invention to possess. However, in another preferred embodiment of the method of the invention, alone or in combination with the other preferred embodiments, the concentration of magnetic nanoparticles is between 1 and 2 mg / mL.

[0100] Likewise, as understood by those skilled in the art, the polyelectrolyte layers, whether at least 2, 3 or 4, may comprise a fluorophore for the purpose of identifying the nanostructure of the invention obtained by the method of the invention. Thus, in a preferred embodiment of the method of the invention, the polyelectrolyte layer of the invention comprises a fluorophore, more preferably, the fluorophore is bonded to PPDA. The term "fluorophore", as well as examples of fluorophore, have been described herein. However, in another even more preferred embodiment of the method of the invention, alone or in combination with the rest of the preferred embodiments, the fluorophore is fluorescein isothiocyanate (FITC).

[0101] After mixing the polystyrene sphere with an aqueous solution of polyelectrolytes as previously indicated, a complex is obtained comprising the polystyrene sphere surrounded by a layer of polyelectrolytes, and said complex is used to carry out step (a) of the method of the invention, that is, the complex is brought into contact with the magnetic nanoparticles to obtain, in this case, the magnetic core of the polystyrene nanostructure of the invention. Once the magnetic core of the nanostructure of the invention has been obtained, step (b) of the method of the invention is carried out.

[0102] Method for obtaining the silica nanostructure of the invention

[0103] In the event that the nanostructure of the invention is based on silica spheres, in a particular embodiment of the method of the invention, step (a) of the method of the invention comprises

[0104] (i) mixing ethanol (EtOH) and water by stirring,

[0105] (i) add to the mixture obtained in (i) ammonia, tetraethoxysilane (TEOS) and at least two magnetic nanoparticles,

[0106] (iii) stir to give rise to the formation of the magnetic core and

[0107] (iv) obtaining the magnetic core formed in (iii) by centrifugation and washing with ethanol.

[0108] Step (i) comprises mixing EtOH with water by stirring. In a preferred embodiment of the method of the invention, said stirring in step (i) is carried out between 1,000 and 1,500 rpm for a time between 2 and 10 minutes.

[0109] Next [step (i)], ammonia, TEOS and at least 2 magnetic nanoparticles are added to the mixture obtained in step (i) and stirred in step (iii). In a particular embodiment of the method of the invention, the stirring in step (iii) is carried out between 500 and 1,000 rpm for 1 to 5 hours.

[0110] Optionally, in another preferred embodiment of the method of the invention, step (i) may comprise the addition of a fluorophore, which in another even more preferred embodiment, said fluorophore is FITO. As those skilled in the art know, the addition of a fluorophore to the mixture causes the nanostructure resulting from applying all the steps of the method of the invention to be fluorescent, and can be identified by fluorescence techniques. The term "fluorophore" and examples thereof have been described in previous paragraphs of the present description.

[0111] Once step (iii) is completed, a magnetic core is obtained, which is then isolated in step (iv) of the method by centrifugation and washing with ethanol. In a preferred embodiment of the method of the invention, the centrifugation in step (iv) is carried out between 1,000 and 5,000 rpm for a time between 2 and 10 minutes. Step (iv) can be carried out as many times as necessary until the magnetic core is isolated with the desired purity. However, in a preferred embodiment of the method of the invention, alone or in combination with the previous preferred embodiments, step (iv) is carried out between 2 and 5 times.

[0112] Once the magnetic core based on a polystyrene sphere or a silica sphere has been obtained, step (b) of the method of the invention is carried out, that is, the magnetic core obtained in step (a) is brought into contact with a culture medium comprising a cell line.

[0113] Any cell culture medium can be used to carry out the method of the invention. Depending on the cell line chosen, those skilled in the art know which is the most appropriate culture medium for the maintenance and growth of said cell line. However, in a preferred embodiment of the method of the invention, the culture medium is selected from the list consisting of DMEM (Dulbecco's Modified Eagle Medium), DMEM / F12, MEM (Minimal Cell Culture Medium), IMDM (Iscove's Modified Dulbecco's Medium), RPMI 1640 (Roswell Park Memorial Institute 1640 medium), McCoy, Ham, BME (Basal Eagle Medium), and EBSS (Earle's Balanced Salt Solution).

[0114] Likewise, any cell line can be used to carry out the method of the invention. The term "cell line" as used in the present invention refers to cells or a population of cells of a single type from a multicellular organism (human, animal or plant) that can be cultured in vitro for extended periods. It is routine practice for a person skilled in the art to choose the type of cell line. Examples of cell lines include, but are not limited to, HEK 293T or HeLa. However, in a preferred embodiment of the method of the invention, alone or in combination with each or all of the previous preferred embodiments, the cell line is HEK293T.

[0115] The selected cell line can express at least one protein ligand of an enveloped virus in its membrane. Cell lines that express at least one protein ligand of an enveloped virus in their membrane can be obtained by genetic engineering. It is routine practice for those skilled in the art to genetically modify a cell so that it expresses in its membrane the desired protein ligand of an enveloped virus, which will be present in the nanostructure of the invention obtained by applying the method of the invention. The terms "enveloped virus" and "protein ligand" have been defined previously in the present description, and their definition is applicable to the method of the invention.

[0116] Thus, in another preferred embodiment of the method of the invention, alone or in combination with each and every one of the previous preferred embodiments, the ligand protein of an enveloped virus is derived from a virus selected from the list consisting of: severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2); influenza virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human immunodeficiency virus (HIV), dengue virus (DENV), and rabies virus. In another even more preferred embodiment of the method of the invention, the ligand protein of an enveloped virus is the spike protein of SARS-CoV-2.

[0117] Once the magnetic core has been brought into contact with the cell line comprising the ligand protein of an enveloped virus in its membrane, step (c) of the method of the invention comprises applying a magnetic field to the cells in the culture medium, thereby forcing the magnetic core into the cell interior. Tools for generating magnetic fields are widely known in the state of the art, and any of them can be applied in the context of the method of the invention. For example, and not limited to, by means of magnets. In a preferred embodiment of the method of the invention, the magnetic core is introduced into the interior of the cells by applying a magnetic field gradient (e.g. 50-70 mT / m) for a time between 2 and 40 hours at a temperature between 30 and 40°C.In another even more preferred embodiment, alone or in combination with the previous preferred embodiments, the magnetic field is generated by placing a magnet at the bottom of the crop.

[0118] After the magnetic nucleus has entered the interior of the cells thanks to the application of the magnetic field, step (d) is performed, which involves the removal of those magnetic nuclei that have not entered the interior of the cells. This removal can be done by washing, centrifuging, or aspirating the culture medium.

[0119] In the event that the elimination of non-internalized magnetic nuclei entails the elimination of the culture medium, then before carrying out step (e) of the method of the invention, a new culture medium is added, that is, a culture medium that does not contain magnetic nuclei.

[0120] Finally, step (e) of the method of the invention comprises applying a magnetic field to the culture medium again to extract the magnetic nuclei from the cell interior, obtaining after extraction a culture medium comprising the nanostructure of the invention. In a manner analogous to step (c), in a preferred embodiment of the method of the invention, the magnetic nucleus is extracted from the interior of the cells by applying a magnetic field for a time between 2 and 40 hours at a temperature between 30 and 40°C. In another even more preferred embodiment, alone or in combination with the previous preferred embodiments, the magnetic field is generated by placing a magnet on top of the culture.

[0121] Optionally, the method of the invention may comprise an additional step (f) after step (e) aimed at isolating the nanostructure of the invention from the culture medium obtained in (e). In a preferred embodiment of the method of the invention, alone or in combination with the previous preferred embodiments, the isolation comprises (i) centrifuging the culture medium for a time between 1 and 10 minutes with stirring between 10,000 and 150,000 rpm, (ii) washing with saline solution, and (iii) filtering. In another even more particular embodiment of the method of the invention, the filtering is carried out by means of a filter comprising a pore size of between 50 and 500 microns.

[0122] DESCRIPTION OF THE FIGURES

[0123] Figure 1. A) Schematic diagram of the synthesis of polystyrene nanoparticles with iron. B) Electron microscopy images of the nanoparticles.

[0124] Figure 2. A) HEK293T cells showing the cytoplasm and SARS-CoV-2 S protein as small dots on the membrane. B) HEK293T cells showing the nucleus and SARS-CoV-2 S protein as small dots on the membrane. NPs inside the cells are seen in brighter dots.

[0125] Figure 3. A) Schematic of the entry and exit of nanoparticles into the cell. 1- entry of nanoparticles crossing the membrane, 2- Nanoparticles in the cytoplasm of the cell, 3- Nanoparticles in the cell cytoplasm close to the membrane, 4- Nanoparticle in the vicinity of the cell membrane about to exit, 5- Nanoparticles leaving the cell coated with cell membrane. B) Electron microscopy images of the nanoparticles. 1- entry of nanoparticles crossing the membrane, 2- Nanoparticles in the cytoplasm of the cell, 3- Nanoparticles in the cell cytoplasm close to the membrane, 4- Nanoparticle in the vicinity of the cell membrane about to exit, 5- Nanoparticles leaving the cell coated with cell membrane.

[0126] Figure 4. Presence of protein S in nanoparticles. A) Schematic of the NPs extracted from cells. B) Electron microscopy image of the NPs extracted from cells. C) Electron microscopy image of the NPs exiting cells. D) Detection of protein S in NPs by immunoblotting.

[0127] Figure 5. Schematic of the polystyrene nanostructure of the invention.

[0128] Figure 6. Schematic of the silicon nanostructure of the invention. EXAMPLES

[0129] Obtaining a nanoparticle with magnetic properties coated with a plasma membrane and a spike protein identical to that of SARS-CoV-2

[0130] Materials and methods

[0131] The synthesis of iron oxide (magnetic) nanoparticles (NPs) is carried out following the method described in Massart et al., Preparation of Aqueous Magnetic Liquids in Alkaline and Acidic Media. IEEE Trans Magn. 1981;17:1247-8. Thus, 1 mL of an aqueous solution (2M) of FeSO^ FW (dissolved in a (2M) HCl solution) and 4 mL of a (1M) FeCls-etW solution are rapidly poured into 50 mL of a NH4OH solution (0.34 M) (29% by weight) with mechanical stirring. Stirring is maintained for 30 minutes and finally the nanoparticles are allowed to settle. After 5 washing cycles (water) of sedimentation and decantation of the supernatant, the iron oxide nanoparticles are dispersed in 50 mL of ultrapure water.

[0132] To produce polystyrene (PS) nanostructures in combination with iron oxide NPs, 130 pL of polystyrene spheres (100 mg / mL) were diluted in ultrapure water. Subsequently, 15 mL of PPDA (1 mg / mL in 0.5 M NaCl solution) was added under gentle ultrasonication and stirred for 1 h. Excess PPDA was removed by 3 cycles of centrifugation in ultrapure water. Then, 15 mL of poly(sodium 4-styrene sulfate) (PSS) (1 mg / mL in 0.5 M NaCl solution) was added and the procedure was repeated until 4 layers of alternating polyelectrolytes were completed, following the layer-by-layer protocol described in Caruso F, Spasova M, Susha A, Giersig M, Caruso PA. Magnetic Nanocomposite Particles and Hollow Spheres Constructed by a Sequential Layering Approach. Chem Mater, American Chemical Society ;13:109-16.

[0133] The PS spheres with four polyelectrolyte layers were dispersed in 10 mL of ultrapure water. Finally, 160 pL of the freshly prepared iron oxide nanoparticle solution was diluted to 5 mL in ultrapure water and mixed with the polyelectrolyte-coated PS solution. The mixture was stirred for 1 h. The resulting colloids were then washed by three centrifugation cycles.

[0134] Additionally, to make the nanostructures fluorescent, a bilayer of PSS (poly(sodium 4-styrene sulfate) and PPDA-FITC and (poly(diallyldimethylammonium chloride) with fluorescein isothiocyanate) was added following the previous procedure.

[0135] Through these procedures, spherical nanoparticles of similar dimensions to the virus (50 to 500 nm in diameter) were obtained, magnetic and fluorescent, of polystyrene nature, favoring the entry and exit of cells using magnetic force and which can be visualized using electron microscopy techniques (Figure 1).

[0136] The HEK293T SARS-CoV-2 spike mutant cell line (Innoprot, P30910) was developed by stably transfecting the SARS-CoV-2 Spike D614G plasmid into the HEK293T cell line, resulting in constant levels of expression of the SARS-CoV-2 spike protein on its cell surface. 50 x 10 3 cells in a 60mm culture dish. This overexpresses the Spike or "S" protein, which gives rise to the crown (typical of the entire coronavirus family) in human cell lines that express the Spike protein.

[0137] After 24 hours, 50 µg of nanoparticles were added to the 60 mm plate, and a 63 mm diameter round magnet (P750 / TCN-63, Supermagnet) was placed directly underneath. This was kept in the cell incubator at 37°C for 24 hours. A further 24 hours were allowed for the nanoparticles to penetrate the cells. After this time, any particles that had not entered the cells were removed by removing the culture medium. Fresh culture medium was added, and this time the magnet was placed on top of the culture plate. This was maintained for 48 hours. All the cell medium containing the nanoparticles was collected and centrifuged for 5 minutes at 13,000 rpm and washed twice with PBS. The cells were then finely filtered through a 200 micron pore size filter to remove any cell debris.

[0138] RESULTS

[0139] The obtained nanoparticles were studied under a transmission electron microscope (Figure 2), where the polyesterene nanoparticle surrounded by iron oxide nanoparticles, with a size between 200-500nm, can be clearly observed.

[0140] Regarding cellular studies, the existence of the S protein in HEK293T cells and the entry of fluorescent nanoparticles into this cell line were first verified. All this was carried out using fluorescence microscopy as shown in Figure 1. Figure 1A shows the HEK293T cells, their cytoplasm, and the SARS-CoV-2 S protein. Figure 1B shows the HEK293T cells, their nucleus, and the SARS-CoV-2 S protein. In addition, the NPs inside the cells can be observed. These fluorescence images demonstrate that HEK293T cells have the S protein in their membrane and also that the synthesized nanoparticles that will be used in the experiment perfectly enter the cells.

[0141] The next step evaluated was to study the trajectory of the NPs inside the cell. As described in the materials and methods section, the NPs are introduced into the cells by placing a magnet at the bottom of the culture dish, so that the NPs will cross the cell membrane and will be located in the cell cytoplasm (Figure 3A, #1, #2 and #3). Once the NPs are inside the cells, a magnet is placed on top, and the nanoparticles exit the cell by forced budding, similar to viral "budding", and coat the membrane that expresses the SARS-CoV-2 S protein (Figure 3A, Figure 4 and Figure 5). To visualize and analyze these processes, electron microscopy images were used. Figure 3B (#1 to #5) shows a representation of these images, where the NPs can be observed in each of the different steps mentioned above.This experimental approach provides information on the internalization of NPs into cells, and the subsequent release of NPs surrounded by the viral S protein, allowing their use in cell interaction studies and viral inhibitor or immunization assays.

[0142] Study of the production of NPs coated with the viral membrane. To demonstrate the existence of the viral membrane around the NPs, and therefore of the synthetic virus (Figure 4A) proposed by this invention, three different studies were carried out. First, the synthetic virus was analyzed by transmission electron microscopy (Figure 4B) and it was observed that the images obtained are quite different from those of the uncoated NPs (Figure 2B), with a clear coating around the NP. The moment in which the NPs leave the cell with the membrane around them was also studied by electron microscopy, as can be clearly seen in Figure 4C. Finally, the presence of the S protein was analyzed by immunoblotting (Figure 4D), a laboratory technique widely used in molecular biology and biochemistry to detect and analyze specific proteins present in a biological sample.

[0143] CONCLUSIONS

[0144] The system is safe, scalable, and easy to manipulate. It has the same size and envelope characteristics as the virus, but lacks infectious capacity (no genetic material). This allows these particles to be used in immunization, viral inhibitor research, or antibody validation, avoiding all the biosafety issues that would be necessary when using viral particles.

[0145] The advantages of this nanobiotechnology-based system are that the particles can be used in: (i) immunization; (ii) studies of compounds for the inhibition of the same penetration mechanisms of viruses; (iii) studies for the selection of antibodies against different viruses; (iv) development of immunodetection kits, among other applications. The versatility of the system is enormous, allowing the expression of different viral ligand proteins (e.g., the Spike protein) and the system to be applicable to any enveloped virus.

Claims

CLAIMS 1. A nanostructure comprising a magnetic core coated by one or more cellular components, wherein said magnetic core comprises magnetic nanoparticles.

2. Nanostructure according to claim 1, wherein the cellular component is a cell membrane, a nucleic acid, or a component of the cell cytoplasm.

3. Nanostructure according to claim 2, wherein the cell membrane comprises at least one protein ligand of an enveloped virus.

4. Nanostructure according to any one of claims 1 to 3, wherein the magnetic nanoparticles are iron oxide nanoparticles.

5. Nanostructure according to any one of claims 1 to 4, wherein the magnetic core comprises at least one polystyrene sphere.

6. Nanostructure according to claim 5, wherein the polystyrene sphere is surrounded by at least two layers of polyelectrolytes.

7. Nanostructure according to claim 6, wherein the polyelectrolyte layer comprises poly(sodium 4-styrene sulfate) (PSS) and / or poly(diallyldimethylammonium chloride) (PPDA).

8. Nanostructure according to claim 7, further comprising a bilayer of PSS and PPDA, wherein PPDA has a fluorochrome attached.

9. Nanostructure according to claim 8, wherein the fluorochrome is fluorescein isothiocyanate (FITC).

10. Nanostructure according to any one of claims 6 to 9, wherein the polyelectrolyte layers are surrounded by the magnetic nanoparticles.

11. Nanostructure according to any one of claims 1 to 4, wherein the magnetic core comprises at least one silica sphere.

12. Nanostructure according to claim 11, wherein the silica sphere comprises the magnetic nanoparticles inside.

13. Nanostructure according to claim 12, wherein the silica sphere comprises at least two embedded magnetic nanoparticles.

14. Nanostructure according to any one of claims 11 to 13, wherein the silica sphere comprises a fluorophore.

15. Nanostructure according to claim 14, wherein the fluorochrome is fluorescein isothiocyanate (FITC).

16. Nanostructure according to any one of claims 3 to 15, wherein the ligand protein of an enveloped virus is derived from a virus selected from the list consisting of: severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2); influenza virus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV-2), human immunodeficiency virus (HIV), dengue virus (DENV), and rabies virus.

17. Nanostructure according to any one of claims 3 to 15, wherein the ligand protein of an enveloped virus is the SARS-CoV-2 spike protein.

18. A composition comprising a nanostructure according to any one of claims 1 to 17, preferably, further comprising a carrier and / or excipient.

19. Composition according to claim 18, wherein the composition is a pharmaceutical composition.

20. Composition according to claim 18 or 19, wherein the vehicle and / or excipient are pharmaceutically acceptable.

21. Composition according to any one of claims 18 to 20, wherein the composition is formulated for parenteral administration.

22. Nanostructure according to any one of claims 1 to 17, or composition according to any one of claims 18 to 21, for use as a medicament.

23. Nanostructure according to any one of claims 1 to 17, or composition according to any one of claims 18 to 21, for use as a vaccine.

24. Use of a nanostructure according to any one of claims 1 to 17, for the in vitro analysis of compounds capable of inhibiting viruses, for the in vitro validation of antibodies or as an in vitro intracellular biopsy system.

25. In vitro method for obtaining a nanostructure according to any one of claims 1 to 17, comprising: (a) contacting magnetic nanoparticles with a support medium to obtain a magnetic core, (b) contacting the magnetic core obtained in (a) with a culture medium comprising a cell line; (c) applying a magnetic field to the cells in the culture medium, thus forcing the magnetic nucleus into the cell; (d) remove those magnetic nuclei that after step (c) have not entered the cells, and (e) applying a magnetic field to the culture medium again to extract the magnetic nuclei from the cell interior, obtaining after the extraction a culture medium comprising a nanostructure according to any one of claims 1 to 17.

26. Method according to claim 25, wherein the magnetic nanoparticles are iron oxide nanoparticles.

27. Method according to claim 25 or 26, wherein the cell line expresses in its cell membrane at least one ligand protein of an enveloped virus.

28. Method according to any of claims 25 to 27, wherein the support means is a polystyrene sphere.

29. Method according to claim 28, wherein prior to step (a), the method comprises mixing at least one polystyrene sphere with an aqueous solution of polyelectrolytes by stirring by sonication with low intensity ultrasonic waves for a time between 0.5 and 4 hours, thus obtaining a complex comprising the polystyrene sphere surrounded by a layer of polyelectrolytes.

30. The method of claim 29, wherein the aqueous polyelectrolyte solution comprises sodium poly-4-styrene sulfate (PSS) and / or poly(diallyldimethylammonium chloride) (PPDA).

31. Method according to claim 29 or 30, wherein the concentration of polyelectrolytes in the aqueous solution is between 0.5 and 3 mg / mL in NaCl between 0.1 and 1 M.

32. Method according to claim 31, wherein the concentration of polyelectrolytes in the aqueous solution is 2 mg / mL in 0.5 M NaCl.

33. Method according to any one of claims 25 to 32, wherein the concentration of magnetic nanoparticles is between 1 to 2 mg / mL.

34. Method according to any one of claims 30 to 33, wherein PPDA has a fluorophore attached.

35. Method according to claim 34, wherein the fluorochrome is fluorescein isothiocyanate (FITC).

36. Method according to any one of claims 25 to 27, wherein step (a) comprises (i) mixing ethanol (EtOH) and water by stirring, (i) add to the mixture obtained in (i) ammonia, tetraethoxysilane (TEOS) and at least two magnetic nanoparticles, (iii) stir to give rise to the formation of the magnetic core and (iv) obtaining the magnetic core formed in (iii) by centrifugation and washing with ethanol.

37. Method according to claim 36, wherein step (i) comprises the addition of a fluorophore.

38. Method according to claim 37, wherein the fluorophore is FITC.

39. Method according to any one of claims 36 to 38, wherein the stirring of step (iii) is carried out between 500 and 1000 rpm for a time of between 1 to 5 hours.

40. Method according to any one of claims 36 to 39, wherein the centrifugation of step (iv) is carried out between 1000 and 5000 rpm for a time between 2 and 10 minutes.

41. Method according to any one of claims 25 to 40, wherein the application of the magnetic field of steps (c) and (e) is carried out for a time between 2 and 48 hours at a temperature between 30 and 40 °C.

42. Method according to any one of claims 25 to 41, wherein the method comprises a step (f) after step (e), which comprises isolating from the culture medium a nanostructure according to any one of claims 1 to 17.

43. Method according to claim 42, wherein the isolation comprises (i) centrifuging the culture medium for a time between 1 and 10 minutes with agitation between 10,000 and 150,000 rpm, (ii) washing with saline solution, and (iii) filtering.

44. Method according to claim 43, wherein the filtering is carried out by means of a filter comprising a pore size of between 50 and 500 microns.

45. Method according to any one of claims 25 to 44, wherein the cell line that expresses in its membrane at least one ligand protein of an enveloped virus is HEK293T.

46. ​​The method of any one of claims 27 to 45, wherein the ligand protein of an enveloped virus is derived from a virus selected from the list consisting of: severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2); influenza virus, herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human immunodeficiency virus (HIV), dengue virus (DENV), and rabies virus.

47. Method according to claim 46, wherein the ligand protein of an enveloped virus is the SARS-CoV-2 spike protein.

48. Method according to any one of claims 25 to 47, wherein the culture medium is selected from the list consisting of DMEM, DMEM / F12, MEM, IMDM, RPMI 1640, McCoy, Ham, BME and EBSS.

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