GB2604128A - Porous silica globules impregnated a denaturing mixture and provided with a phospholipid mantle placed in an aqueous suspension to capture and dissolve - Google Patents

Abstract

Porous silica globules impregnated with a denaturing mixture and provided with a phospholipid mantle placed in an aqueous suspension in order to capture and dissolve airborne viruses. The porous silica core 1 is impregnated with a denaturing mixture to break down the encapsulated protein capsids and virus nuclei. The covering mantle 3 comprises a first layer 6 composed of a mixture of phosphatidylcholine and phosphate-diethanolamine and a second layer 5 comprising phosphatidylcholine and phosphatidylserine. The second layer has inserted cholesterol nanogranules 4 comprising linoleic acid, sialic acid ganglioside GM1, carnosic acid and tetramethylenediamine (TEMED). The denaturing mixture impregnated into the silica particles comprises water, dimethyl sulfoxide, guanidinium thiocyanate, guanidinium chloride, urea, sodium dodecyl sulphate, ammonium persulphate, propidium iodide, thiazole orange and fluorescein isothiocyanate. The globular suspension can be applied to environmental sanitation systems that treat the air for pathogen abatement, also with the complementary use of UV radiation. In particular, they can be used in aerosol spraying systems to counteract influenza viruses.

Description

Porous silica globules impregnated a denaturing mixture and provided with a phospholipid mantle placed in an aqueous suspension to capture and dissolve airborne viruses

DESCRIPTION

Virus decontamination of solid surfaces -even large ones -is relatively simple using proven techniques such as solvents, soaps, high-temperature vapours and can be carried out in various ways. On the other hand, the pathogenic suppression of the air for considerable volumes and over time is rather problematic, especially for air treatment systems with aerosols. Although they afford maximum viral removal capacity, there is still the problem of eliminating the vapour in the equipment without releasing residues of irritating substances.

In order to overcome the criticality of such environmental sanitation systems, a technological system that simulates cellular mimesis to facilitate the capturing and dissolution of viral particles has been developed For the latest generation of aerosol spraying systems, in particular those using electro-sprays in combination with UV radiation, the use of the aqueous suspension of micro-globules has been verified. This does not release any trace of volatile irritant substance into the environment being treated. Optimum performance, also in terms of safety of the result, is determined by the high concentration of antiviral mixtures in the core of the globules. These are carefully sealed against the aqueous suspension by a cell-like lipid membrane.

The suspension captures viruses already in the aerosol phase and ensures condensation, precipitation, and separation from the treated air. Viral dissolution continues even when the globules are precipitated from the aerosol and collected in the reservoir to return them to the circulation.

The globules covered by this patent application are composed of a central core of amorphous silica with an average diameter "43.," of about 10 jim (Figure 1). On the surface of these, a mantle composed of a phospholipid bilayer is deposited.

The amorphous silica core presents a diffuse porosity which is impregnated by a mixture of compounds consisting of (by weight) water H2O (40%), dimethyl sulphoxide C2H6OS (30%), guanidinium thiocyanate C2H6N4S (10%), guanidinium chloride C1-16CIN3 (6%), urea CH4N20 (5%), sodium dodecyl sulphate C12H25NaSO4 (4%), ammonium persulphate (NH4)25208 (3%), propridium iodide C27H3412N4 (1.1%), thiazole orange C26H24N203S2 (0.6%), and fluorescein Isothiocyanate C21H11N06S (0.3%). The function of the mixture components is to act as protein denaturants, nucleic acid denaturants, and fluorescent tracers of dissolved products.

In particular, the compounds guanidinium thiocyanate, guanidinium chloride, and sodium dodecyl sulphate are able to break disulphide bridges in viral capsid proteins, deforming their tertiary structure and allowing them to be more easily dissolved by the solvent dimethyl sulphoxide.

Fig. 1 shows the amorphous silica core (1) with the surface cavities (2) containing the mixture in aqueous form. The amorphous silica core (1) is covered by the lipid mantle (3), which has lipid rafts with viral receptors for virus attachment (4).

The mantle, shown in more detail in the enlargement in Fig. 2, is formed by a lipid bilayer of phospholipids (3) with a different composition. The first layer adheres to the silica core (6), and the second is in contact with the external environment (5). The polar heads of the phospholipids in the first layer (5) of the mantle (3) are oriented towards the amorphous silica core (1), while those in the second layer (6) are oriented outwards as shown in Fig. 2.

The first layer (6) is composed of a mixture of 60% phosphatidylcholine and 40% phosphate-diethanolamine (w/w) in order to ensure high mobility and deformation capacity and adaptation of the membrane to the geometry of the amorphous silica core (1).

The second layer (5) is composed of a mixture of 95% phosphatidylcholine and 5% phosphatidylserine (w/w), making it more rigid and viscous and able to provide resistance to environmental stress from outside the globule.

The second phosphatidylcholine layer (5) contains nanogranules (4) consisting of 70% cholesterol, 18% linoleic acid C28H3202, 10% sialic acid ganglioside GM1, 1.5% carnosic acid C20E12804, and 0.5% tetramethylethylenediamine (TEMED) C61-116%.

The nanogranules (4) inserted into the second lipid layer (6) create lipid rafts that are positioned near the cavities (2) of the amorphous silica core (1).

The nanogranules (4) that make up the lipid rafts perform the dual function of viral receptor and virus entry port (10). This is the core of the cellular mimesis function performed by the globule as shown in the sequences in Fig. 3.

In the first sequence of Fig. 3, the virus is close to a globule. The external protein hemagglutinin HA (8) of the viral pericapsid (9), common to all influenza airborne viruses, latches onto the sialic acid end of the ganglioside (7) contained in the cholesterol nanogranule of the lipid raft, thereby allowing local deformation of the globular layer and fusion of the phospholipid membrane (3) with the viral pericapsid (9).

The residue of the lipid fusion of the viral pericapsis with cholesterol and the globular phospholipid membrane (12) is shown in the second sequence of Fig. 3.

The subsequent access of the viral particle into the underlying cavity of the porous silica core (1) is depicted in the second progressive sequence of Fig. 3.

The virus encapsulated in the porous silica cavity (2) underlying the residue of the lipid raft (12) undergoes the aggression of the substances impregnated in the core as shown in the third sequence of Fig. 3.

After denaturation of the protein capsid (when it has dissolved) the specific compounds in the mixture can gain access to the viral nucleic acids (11), whether DNA or RNA, by intercalating between the bases of the ribose chain, fragmenting them, and rendering them inactive and unable to infect as shown (12) in the fourth sequence in Fig. 3.

The action of altering and breaking down nucleic acids can be greatly accelerated by the use of UV light, which triggers the formation of free radicals and peroxides through ammonium persulphate (NH4)2S208contained in the impregnation mixture. Particularly effective is the introduction of small amounts of carnosic acid C20F12804 into the cholesterol granules. This performs a dual function by preserving cholesterol and linoleic acid from oxidation and above all promoting the fusion of the viral lipid membrane to that of the globule, thus mimicking the dynamics of viral penetration into the pulmonary alveoli. When an influenza virus penetrates lung tissue, haemagglutinin HA binds to the sialic acid of ganglioside receptors, and the alveoli respond with inflammation, acidifying the lungs. Paradoxically, acidification of the lungs favours virus penetration because haemagglutinin folds in an acidic environment and promotes the adhesion of the viral lipid pericapsid to the cell membrane. Thus, the more inflamed and acidic a lung is, the more a flu virus can infect it because lung acidity is effective only against bacterial pathogens and not viral ones. The insertion of carnosic acid into the cholesterol granule promotes lipid membrane fusion by exploiting the folding of viral haemagglutinin HA.

Each globule of porous silica, the subject of this discovery, has an extraordinary capacity for surface capture. This is due to the size (i.e. that of viruses with an average diameter more than 100 times smaller). It has been calculated that each porous silica globule has the potential to capture 30,000 viral particles with an average diameter of 100 nm because each viral particle engages a surface area of approximately 0.01 km2 and a porous silica globule (10 km in diameter) has a mantle area of over 300 km2.

Considering that 1 ml of aqueous suspension can contain approximately one billion globules of porous silica, it can be deduced that a bottle with a volume of 100 ml can contain as many as one hundred billion globules of porous silica with a capture potential of 3 million billion viral particles.

In a highly pathogen-contaminated airborne environment, a concentration of up to 50 billion viral particles per cubic metre of air can occur. As a result, using a small volume of aqueous suspension, huge amounts of air can be purified for a long time before replacing the globular suspension, thus making this decontamination technology highly efficient and cost-effective.

The globular suspension is obtained in six sequential steps as shown in Fig. 4 and is based on the property of porous silica to absorb at temperatures around 60°C a quantity of liquid mixture of saturation approximately 25% higher than the quantity of saturation at room temperature of 20°C. During cooling, a part of the mixture is desorbed and fills the surface cavities of the porous silica because of the equilibrium of the pressure inside the globule with respect to the outside. This results in a pseudo-spherical conformation of the lipid mantle (3).

In the first phase (13) Fig. 4, the porous silica globules are impregnated at 60°C with the mixture (dissolving denaturing solution) for proteins and nucleic acids, thus bringing the globules to saturation.

In the second phase (14) Fig. 4, the first lipid mantle is created on the porous silica globules by introducing them into a mixture of phosphatidylcholine and phosphatediethanolamine with the globules in excess of the lipids and maintaining the temperature at around 55°C so that the polar heads of the uniform phospholipids adhere around the globule until the porous surface cavities are partially filled.

The third step (15) (Fig. 4), consists in the preparation of the nanogranules of cholesterol and sialic acid gangliosides by inserting their homogeneous mixture in a dissolver with water until a size of about 200 nm is obtained.

In the fourth step (16) (Fig. 4), the nanogranules are mixed with the first layer of globular mantle so that they are evenly distributed on the mantle and are sufficiently spaced from each other. The nanogranules will preferentially be positioned within the cavities of the impregnated porous silica and partially filled by the phospholipids of the first phase because in these recesses, they are able to have more contact surface to form lipid bonds. The operation should be carried out at around 50°C so that the lipids remain fluid and more than 80% of the excess impregnation mixture is retained on the porous silica.

In the fifth phase (17) Fig. 4, the second lipid mantle formed by the phosphatidylcholine and phosphatidylserine is formed by inserting this into the globular suspension so that the mantle is uniformly coated at a temperature of 45°C.

The phospholipids of the second layer will be arranged with the polar heads outwards and the lipid tails attached to the first mantle.

In the sixth and final preparatory step (18) (Fig. 4) the globules are cooled to ambient temperature (approx. 20°C) so that the porous silica globules release by desorption the excess mixture. This will fill the surface cavities, thereby pushing the first layer of phospholipids and the cholesterol nanogranules outwards.

Swellings form on the globular surface leaving the cholesterol nanogranules with their viral receptors partially uncovered (7). Lipid rafts form on the outside of the lipid bilayer, and cavities are filled with dissolving denaturing mixture in the area below; magnification of the illustration (2X) (Fig. 4).

The globules formed by this serial process are suitable for the capture and dissolution of suspended pathogens.

The intrinsic properties of this particular globular suspension allow for a high ratio between the volumetric flow rate of the air being treated and the mass of the globules in suspension. This achieves an exceptional compactness factor with the use of modest quantities of the viral dissolution preparation.

As an example, for a treatment plant with 500-1000 rnVh of air, a bottle of 100200 ml of globular suspension is sufficient to guarantee decontamination from viral pathogens for 8-10 weeks of continuous 24-hour sanitisation.

Thiazole orange C26F124N20352 was also included in the impregnation mixture propriodine Iodide C27H3412N4 and acts as a fluorescent dye when specifically intercalated with nucleic acids. Fluorescein isothiocyanate C21FI11N055 binds to the protein and phosphatidylserine portion of the outer mantle layer so that the globules acquire a fluorescence intensity directly proportional to the viral load adsorbed and dissolved.

In particular, after viral adsorption the fluorescein isothiocyanate C22H22N06S also penetrates the cholesterol nanogranule around the viral pericapside residues and attaches to the fostatidylserine present in this, thereby creating a fluorescent halo.

Fluorescent dyes reveal when the dispersion needs to be replaced because the globules are coloured orange when exposed to sunlight and become extremely fluorescent when subjected to UV light.

By exploiting this fluorescent property, it is also possible to equip environmental sanitation systems, which use the globular suspension that is the subject of the present invention, with an automatic system in order to monitor the exhaustion of the capture capacity (subject of a separate patent application). Detection of the degree of saturation of the suspension enables the automatic filtering of the globules to be included so that the use of the equipment can be optimised according to the pathogen load present in the environment where it operates. A small percentage of ammonium persulphate (NH4)25208 has also been added to the impregnation; the function of this is to peroxidise and polymerise the linolenic acid with the cholesterol present in the granule; this polymerisation occurs after virus capture and dissolution and is essential to prevent the cholesterol granule from being dissolved by the dimethyl sulfoxide C2H6OS present in the impregnation mixture and from crossing the phospholipid membrane and spilling outside the globular suspension. To catalyse the cleavage of ammonium persulphate (NH4)2S208, so as to accelerate the peroxidative polymerisation reaction, a small percentage of tetramethylethylenediamine (TEMED) C6H26N2is added to the granule together with linoleic acid C281-13202.

With the polymerisation of linolenic acid and cholesterol, a facsimile 'scarring' of the passageway that the virus opened during fusion and penetration into the globular phospholipid membrane takes place.

The suspension that causes the pathogenic structures to break down through the unique mechanism of cell mimesis does not result in the release of reaction products into the environment, even after prolonged operation.

Upon exhaustion, the suspension is collected in a special filtration capsule located within the environmental sanitation system, which collects the spent globules. Once removed from the environmental sanitation system, this capsule is not a special waste because the products it contains (i.e. aggregates of amino acids, lipids, and ribose residues) are biologically inactive. The solution of the aqueous suspension with the globules contained in the refill bottles is brought to saturation with a citric acid solution (40% by weight) in order to guarantee the preservation of the product and preserve the oxidation of the globules during operation in the environmental sanitisation system, thus resulting in an excellent anti-scale action if hard water is used for periodic refilling. Citric acid also acts as both an antibacterial agent and an acidifier of the environment outside the globules; this promotes viral capture and fusion of globular lipid membranes with viral pericapsids.

Users of the specific environmental sanitisation systems, which employ this particular globular suspension technology covered by this invention, will not have any problems to deal with. The bottle containing the globular suspension and the filter cartridge need only be replaced periodically.

The industrial manufacture of this innovative and high-performance suspension, which uses commercially available materials, is cost effective and does not have a detrimental effect on operating costs. It can therefore be mass-produced to cover a very high diffusivity.

Claims (7)

1. CLAIMS1) Porous silica globules impregnated with an aqueous mixture and covered with a mantle consisting of a phospholipid bilayer and placed in an aqueous suspension with 5 the function of capturing and dissolving airborne viruses for application in environmental sanitation systems including: (a) a covering mantle consisting of a first layer with the respective polar heads oriented towards the porous silica, composed of a mixture of (by weight) 60% phosphatidylcholine and 40% phosphate-diethanolamine and a second layer with the respective polar heads facing outwards. Inserted in these outward facing polar heads cholesterol nanogranules overlying the superficial porous cavities of the globule and equipped with viral receptors in contact with the aqueous suspension outside the globules, which is composed of a mixture of (by weight) 95% phosphatidylcholine and 5% phosphatidylserine b) nanogranules composed of 70% cholesterol, 18% linoleic acid C281-13202, 10% sialic acid ganglioside GM1, 1.5% carnosic acid C20H2804, and 0.5% tetramethylethylenediamine (TEMED) C6I-126N2 inserted into the second layer consisting of a mixture of (by weight) 95% phosphatidylcholine and 5% phosphatidylserine c) An impregnation mixture consisting of (by weight) water H20 (40%), dimethyl sulphoxide C21-160S (30%), guanidinium thiocyanate C2H6N4S (10%), guanidinium chloride CH6CIN3 (6%), urea CH4N20 (5%), sodium dodecyl sulphate C12H25NaSO4 (4%), ammonium persulphate (NH4)2S208 (3%), propridium iodide C22F13412N4 (1.1%), Thiazole Orange C26H24N203S2 (0.6%), and fluorescein Isothiocyanate C22H22NO5S (0.3%).
2. (d) An aqueous mixture constituting the liquid suspension for storage and distribution of the globules, consisting of (by weight) 60% water H20 and 40% citric acid C6H802 2) Porous silica globules as per claim 1 with a of 1-100 lam 2.1) Porous silica globules as per claim 2 with a suggested diameter of 10 km 3) Porous silica globules according to claim 1 characterised by porous surface cavities with a depth of 10-1000 nm.
3. 3.1) Porous silica globules as per claim 3 of which the suggested depth is 100 nm.
4. 4) Porous silica globules according to claim 1 wherein the mechanism of action of the globular suspension against airborne viruses takes place according to a sequential process of capture by sialic receptors (7) on cholesterol nanogranules (4), fusion of the lipid membrane (3) with the viral pericapsis (9), incorporation of the virus (10) into the surface cavity (2) of the porous silica (1), denaturation and dissolution of the virus protein capsid (10), intercalation of the nucleic acids, and disruption of the ribose sugar phosphate chains.
5. 5) Porous silica globules according to claim 1 wherein the mechanism of action of interrupting the outward outflow of the imbibition mixture from the interstitium ofSthe lipid membrane (3) left by the englobed and dissolved virus according to claim 4 carried out by polymerisation activated by peroxidation of linolenic acid contained in the cholesterol nanogranule (4) by ammonium persulfate (NH4)2520scontained in the impregnation mixture and catalysed by tetramethylethylenediamine (TEMED) C6F116N2 contained in the nanogranule (4).
6. 6) Porous silica globules as per claim 1 wherein the mechanism of action of the fluorescence after viral incorporation performed by propridium iodide C27F134I2N14 together with thiazole orange C26H24N20352 that intercalate with the nucleic acids in the cavity and do not cross the membrane (3) and fluorescein isothiocyanate C21l-I11NO5S that permeates the membrane (3) during pericapsis fusion (9) binding to viral protein residues and to phosphatidylserine of the outer mantle layer (3).
7. 7) Porous silica globules according to claim 1 wherein the obtaining of the globular suspension is carried out in 6 distinct serial steps: * the impregnation (13) at 60°C of the porous silica globules (1) with the mixture of claim 1 paragraph d) * the forming (14) of the first layer (5) of the mantle (3) at 55°C with the mixture of claim 1 paragraph c) * the preparation (15) of the nanogranules of cholesterol (4) and sialic acid gangliosides (7) with the mixture of claim 1 paragraph 3 with dissolution in water until the size of 200 nm is obtained * the insertion (16) of the cholesterol nanogranules (4) conducted at 50°C with the dissolution obtained from the previous third phase (15) * the formation (17) of the second layer (6) of the coat (3) performed at 45°C with the mixture of claim 1 paragraph c) * the cooling (18) of the dissolution at 20°C