WO2018015466A1 - Virucidal metallic nanoparticles and uses thereof - Google Patents

Abstract

The invention relates to virucidal metallic nanoparticles, virucidal compositions comprising thereof and uses thereof in treatment and/or prevention of viral infections, for sterilizations and for disinfections.

Description

VIRUCIDAL METALLIC NANOPARTICLES AND USES THEREOF

Field of the Invention

The invention relates to virucidal metallic nanoparticles, virucidal compositions comprising thereof and uses thereof in treatment and/or prevention of viral infections, for sterilizations and for disinfections.

Background of the Invention

Infectious diseases account for ~20% of global mortality, with viral diseases responsible for about one third of these deaths. Lower respiratory infections and Human Immunodeficiency Virus (HIV) are among the first ten causes of deaths worldwide, and they contribute substantially to health care costs. The best approach to prevent viral infections is vaccination, however vaccines are not always available. Additionally, a higher incidence of some viral infections is found in underdeveloped countries where the healthcare system struggles to provide sufficient vaccine coverage. Importantly, after infection, the only treatment option available is to provide drugs to support the immune system in the fight against the infection.

Current antiviral therapeutics can be subdivided into small molecules, proteins able to stimulate the immune response (e.g. interferon), or oligonucleotides. These therapies are mainly directed against HIV, Hepatitis B Virus (HBV), Hepatitis C Vims (HCV), Human Cytomegalovirus (HCMV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VZV), and influenza virus. It should be stressed that specific antiviral treatments are lacking for the majority of viruses. Current therapeutic approaches mainly act intracellularly on viral enzymes that are essential for viral replication but differ from any other host enzyme to allow for selectivity. However, since viruses largely depend on the biosynthetic machinery of the infected cells for their replication, the specificity of antiviral drugs is often far from ideal resulting in a general intrinsic toxicity associated with such treatment. Furthermore, most viruses mutate rapidly due to error prone replication machinery, therefore they often develop resistance to antiviralsS. Finally, the use of virus specific proteins as a target of antiviral drags makes it difficult to develop broad-spectrum antivirals acting on a large number of viruses that are phylogenetically unrelated and structurally different (e.g. viruses with or without an outer lipid envelope). It can be reasoned that the ideal viral drag is a broad-spectrum non-toxic material that acts outside the host and irreversibly inhibits viruses, i.e. a virucidal drug.

drugs that are active outside the cellular environment (such as the ones described above) currently only display virastatic activity, i.e. an inhibition due to their binding to the host cell receptors or the viral ligands, leading to reduced cell-virus interactions. Unfortunately, virastatic activity depends on a binding event and hence is reversible. As such, these drugs are inadequate for medical applications, as upon dilution the drug is released from the cell or virus, and no permanent inactivation occurs, allowing the virus to once again infect. The only three polyanionic anti-HIV-1 microbicides that reached phase III clinical trial (i.e. polysulfonated PRO2000, the polysulfated Carraguard, and cellulose sulfate Ushercell) did not prevent the vaginal HIV-1 transmission and even increased the rate of infection. One of the possible explanations is that their effect was simply virastatic and hence vaginal and seminal fluids lead to the dilution of both the viruses and the active compounds, which resulted in the complete loss of binding and active virus release. It should be stressed that virucidal drugs have irreversible effects on the virus; indeed their effect is retained even if dilution occurs after the initial interaction with the virus. This property is the key distinguisher from virastatic drugs.

The challenge is to find virucidal nanoparticles that have minimal side effects on the host, and are thus able to act as virucidal drugs possibly in a broad-spectrum way. As for the polyanionic anti-HIV-1 drags another possible explanation for their failure is that their were toxic (generating local lesions) before being virucidal. There is a vast literature on many virucidal materials ranging from simple detergents, to strong acids, or more refined polymers, and nanoparticles in some cases capable of releasing ions are known virucidal molecules. In all of these cases, the approaches utilized to irreversibly inhibit the virus have intrinsic cellular toxicity side effects. Currently, there is no approved drug that shows virucidal activity. The present invention was able to solve this problem by providing virucidal metallic nanoparticles having unique properties.

Summary of the Invention

In one aspect, the invention provides a virucidal metallic nanoparticle comprising multiple alkyl sulfonate ligands that provide the attachement receptor for viruses, and optionally multiple additional alkyl ligands, wherein

the alkyl sulfonate ligand is - and wherein

Z is absent or selected from the group comprising O, S, and

y is 5 to 20, X is substituted or unsubstituted cyclo-C₅-C₇-alkyl or aryl, wherein one or more substituents are selected from the group comprising C₁-C₄-alkyl and -OH. and the optional additional alkyl ligand is -Q-CH₂-(CH₂)w, and wherein

Q is absent or selected from O or S,

w is 4 to 20.

In a further aspect, the invention provides a pharmaceutical composition comprising an effective amount of one or more virucidal metallic nanoparticles of the invention and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

In another aspect, the invention provides the virucidal metallic nanoparticle of the invention for use in treating and/or preventing viral infections and/or diseases associated with viruses.

In another aspect, the invention provides a virucidal composition comprising an effective amount of the virucidal metallic nanoparticles of the invention and optionally at least one suitable carrier.

In another aspect, the invention provides a method of disinfection and/or sterilization comprising using the virucidal composition of the invention or the virucidal composition of the invention.

In another aspect, the invention provides a device comprising the virucidal composition of the invention or the virucidal metallic nanoparticles of the invention and means for applying or dispensing the virucidal composition or the virucidal metallic nanoparticles.

In another aspect, the invention provides a use of the virucidal metallic nanoparticles of the invention or the virucidal composition of the invention for sterilization and/or for disinfection.

Brief description of figures

Figure 1 shows Virucidal Experiments. A) Heparin (top) MES-NPs (middle) and MUS:OT-NPs (bottom) viral infectivity curves (left) and virucidal assays (right). The percentages of infection were calculated comparing the number of plaques in treated and untreated wells. The results are mean and standard error of the mean (SEM). B) Virucidal activity of MUS coated NPs against HPV 16, RSV, LV-VSV-G, and DENV-2 viruses. DENV-2 virucidal test is performed against MUS-NPs all others against MUS:OT-NPs. 10⁴ plaque forming units, pfu of RSV or focus forming units, 10⁴ ffu of HPV were incubated with 100 μg/mL of MUS:OT-NPs for 2 hours at 37°C. In case of LV-VSV-G, J O⁶ TU/ml were incubated with 1000 μg/mL of MUS.OT NPs for 2 hours at 37°C. In case on DENV2 80954 infectious units (IU) were incubated with 200 μg/mL of MUS-NPs. The mixtures were then titrated onto cells and the residual infectivity was determined at dilutions at which the NPs are no longer active. The results are the mean and standard median error (SEM) of 3 independent experiments performed in triplicate. *** p<0.001 C) MUS:OT- NPs inhibition of viral infectivity against HSV-2 versus time. HSV-2 viruses ( 10⁵ plaque forming units, pfu) were pre-incubated at 37°C with the same concentration of MUS:OT-NPs (100 μg/mL) for the times indicated and the mixtures were subsequently titrated on Vero cells at different time points. The infectivity was evaluated after 24 h, only at the dilutions where NPs were no longer active in the inhibition assay. Results are the mean of 3 independent experiments performed in triplicate and are expressed as mean and SEM.

Figure 2 (A) shows Electron Microscopy Studies of Nanoparticles-Virus Interactions. A) HSV-2 and its association with MUS:OT-NPs. The samples were imaged with cryo-TEM analysis or the samples were stained with uranyl acetate 0.5% for 5 min and images acquired by TEM. The scale bars are 100 nm. B) % distribution of MUS:OT-NPs associated with HSV-2 over time determined by analysing between 50 and 100 cryo-TEM images per condition. In the panel below images representative the different conditions are shown.

Figure 3 shows Molecular Dynamics Simulations. A) Top view of a small sulfonated gold NP (2.4 nm core) selectively binding to HPV capsid L1 protein pentamer, after 25 ns of simulations. Red and yellow spheres show negatively charged terminal sulfonate groups of the MUS NP. Positively charged HSPG-binding residues of L I (K278, K356, K361 , K54 and K59) are shown in blue. Inset highlights the strong selective coupling between sulfonate groups and HSPG- binding residues (K356, K361 , K54 and K59).B) Side view of a large gold NP (5 nm core) binding to two L1 pentamers at initial time (small representation, top left) and after 10 ns simulations (main representation). Red arrows indicate the reorientations of L1 pentamers. C) Schematic diagram illustrates how strong multi-site binding of NPs to HSPG-binding residues can induce irreversible changes in the arrangement of L1 capsid proteins. Figure 4 shows Characterisation of MUS:OT-NPs (A) TEM images of the MUS:OT gold nanoparticle cores. Scale bar 50 nm. (B) Particle size distribution obtained from TEM analysis: the average core diameter was 2.8±0.6nm.

Figure 5 shows Characterisation of MUS:OT-NPs ligand composition. Ή-NMR (400 MHz, Broker A VANCE 400) of MUS:OT gold nanoparticles etched with Iodine solution in MeOD-d4 reveals 34% OT content, i.e. a 2: 1 MUS:OT stoichiometric ratio.

Figure 6 shows Characterisation of MUS-NPs (A) TEM images of the MUS gold nanoparticle cores. Scale bar 50 nm. (B) Particle size distribution obtained from TEM analysis: the average core diameter was 2.5±0.7nm.

Figure 7 shows Characterisation of EG2-OH-NPS A) TEM images of the EG2-OH gold nanoparticle cores. Scale bar 50 nm. B) Particle size distribution obtained from TEM analysis: the average core diameter was 6.²⁰.8 nm.

Figure 8 shows Characterisation of MUP-NPs A) TEM images of the MUP gold nanoparticle cores. Scale bar 50 nm. B) Particle size distribution obtained from TEM analysis: indicating a bimodal distribution of particle sizes. One population of NPs is cantered at 3.5nm. Nanoparticles with a diameter less than 0.5 nm were below the threshold of the data analysis used.

Figure 9 shwos Characterisation of MES-NPs A) TEM images of the MES nanoparticles. Scale bar 50 nm. B) Histogram of particle size distribution shows particles having mean core diameter around 2.6 nm. Threshold is set to 0.6 nm for particle counts.

Figure 10 shows Characterisation of DOS-NPs TEM images of the DOS iron oxide nanoparticle cores. Scale bar 25 nm

Figure 11 shows Differential inhibitory activity of MUS:OT-NPs against Adeno Associated Viruses. Confocal Laser Scanning Microscopy (CLSM) images AAVs (AAV2; AAV5) with MUS:OT NPs. Sulfonated NPs, used at 100 μg/mL (100 nM) and the virus was incubated for Ih at 37°C prior to addition to CHO-K1 cells. Transduction efficiency in terms of GFP+ cells was evaluated by CLSM. Transmitted light (upper panels) and green (GFP+, lower panels) channel are shown. Virus without NPs was used as a control.

Figure 12 shows Attachment assays for MUS:OT-NPs and Heparin. MUS:OT NPs and Heparin were incubated at different concentrations with I lSV-2 (MOI 0.005) for 2 hours at 4°C this condition allows viral binding but not entry to the cells. After the 2 hours the inoculum was removed and after extensive washing with PBS and finally MEM containing MTC was added. The percentage of infection were calculated comparing the number of plaques in treated and untreated wells. The EC50 was calculated with Prism software. These data are the results of 3 independent experiments performed in duplicate. Figure 13 shows MUS:OT-NPs inhibition of infection as a function of incubation time. MUS:OT was incubated at different concentrations with HSV-2 (MOI 0.001) for 0 hours or 1 hour at 37°C and then added on cells. After the 2 hours of infection at 37°C the inoculum was removed and MEM containing MTC was added. The percentage of infection were calculated comparing the number of plaques in treated and untreated wells. The EC50 was calculated with Prism software. These data are the results of 3 independent experiments performed in duplicate. Figure 14 shows Representative cryo-TEM images of HSV-2 and MUS:OT-NPs. The left image shows three HSV-2 viruses. For the purpose of counting the number of NPs per vims in this image was two 5-20 NPs (bottom) and one covered (top). The right hand image shows two HSV- 2 viruses. These were counted as one 5-20 and one covered.

Figure 15 shows MUS:OT NPs associated with HPV- 16 PsVs. The upper panel the images were acquired by cryo-TEM, In the panel below the samples were stained with uranyl acetate 0.5% for 5 min and acquired by TEM. Images a and d represent the HPV- 16, while b, c, e and f represent different types of NPs association with HPV- 16 PsVs. The scale bars indicate l OOnm.

Figure 16 shows Virucidal Assays and Interaction with HSV-2 viruses for EG2-OH-NPS. A)

Cryo-TEM (up) and negative staining TEM (down) images of HSV-2 viras (105 pfu) treated with EG2-OH NPs. B) Virucidal assay of HSV-2 viras treated with EG2-OH NPs. C) % distribution of not sulfonated EG2-OH Au-NPs associated with HSV-2 after 1.5 h of incubation. The majority of viruses were not associated with any nanoparticle. The distribution was calculated analysing between 50 and 100 images.

Figure 17 shows Gel Electrophoresis for purified LV-VSV-G incubated with MUS:OT NPs. A) Agarose gel stained with Instant Blue™ (Expedeon). Purified LV-VSV-G incubated with NPs (lane 2, LV + MUS:OT) was run in parallel with lentiviras alone (lane 1, LV) and MUS:OT NPs (lane 3, MUS.OT) on agarose gel (0.8 wt %). B) Agarose gel stained with GelRed (Biotium). Purified LV-VSV-G incubated with NPs (lane 2, LV + MUS:OT) was run in parallel with lentiviras alone (lane 1, LV) and MUS:OT NPs (lane 3, MUS:OT) on an agarose gel (0.8 wt%). Image was acquired under UV lamp.

Figure 18 shows Structure of the HPV capsid segment. (A, B) Electrostatic potential surfaces of 3 L1 protein pentamers of HPV capsid. The light pink surface is negatively charged, while the light blue surface is positively charged. The equipotential value of positive surface is 7.5V, negative surface is -7.5V. Amino acid residues shown in blue are implicated in HSPG binding. C) Strong interactions of large NP (5 nm core) with the bottom side of the L1 pentamers (10 ns of simulation). Scale bars are 1 nm. Figure 19 shows Structure of the dengue virus. A) Whole capsid of a dengue vims. Residues shown in blue are the HSPG binding sites (K305, K307, K310, K295, K291 , R288, R286, K284, R188, K388, K393 and K394). Red circle shows the high concentration of HSPG binding sites.

Scale bar is 10 nm. B) Top view of five envelope protein E units with HSPG-binding residues (blue). Scale bar is 1 nm. C) Side view of the NP interacting with HSPG binding sites after 10 ns of simulations, (inset) a magnified view of the coupling. Scale bar is 1 nm.

Figure 20 shows Structure of the dengue virus. A) Whole capsid of dengue virus. Scale bar is 10 nm (B, C) Electrostatic properties of the capsid segment of the dengue virus. Electrostatic equipotential surfaces of this capsid segment are shown in pink (negative potential, -2.3 V) or light blue (positive potential, 2.3 V). Scale bar is 1 nm. Panels B and C show the top view and the side view of the capsid segment, respectively.

Figure 21 shows Post treatment assay. HSV-2 (MOI 0.01) was added to cells for 2h at 37°C. After the removal of the viral inoculum different concentrations of MUS:OT-NPs were added to the infected cells. Results are expressed as % of viral titer, calculated in comparison with untreated wells. Results are mean of 3 independent experiments performed in triplicate.

Figure 22 shows Virucidal assay for DOS-NPs against LV-VSV-G. LV-VSV-G virucidal assay with sulfonated iron oxide NPs, DOS (500 μg/mL), and untreated LV-VSV-G virus after 2h at 37°C. The mixture of LV-VSV-G and NPs were then titrated onto the cells and the residual infectivity was determined at dilutions at which the NPs are no longer active. LV-VSV-G was used as a control. The transducing unit/mL (TU/mL) of LV-VSV-G vims with and without NPs were measured after 48h post-infection.

Figure 23 shows Dose response and viracidal activity of MUS-NPs against Dengue virus type 2.

A) MUS-NPs were incubated at different concentrations with DENV-2 (MOI 0.03) for 1 h at 37 °C and then were added to Vero cells. After 3 days incubation fluorescent immuno-stained plaque assay was performed. The % of infection were calculated comparing the numbers of infected cells in treated and untreated wells using Image! software. The EC50 was calculated using GraphPad Prism. The results are mean and sem from experiment performed in triplicate.

B) Viracidal assays were performed by incubating DENV-2 (80954 IU) with 200 μg/mL of MUS-NPs for 2 h. The viracidal effect was determined by titrating serial dilutions of the mixtures on Vero cells and the residual infectivity was assessed at dilutions at which the nanoparticles are no more effective. The results are mean and sem of 3 independent experiments. Figure 24 shows gold nanoparticules ligand synthesis and iron oxide nanoparticules ligand synthesis.

Figure 25 shows dose response. Figure 26 shows virucidal effect of small highly sulfonated metallic nanoparticles.

Detailed description of the Invention

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In the case of conflict, the present specification, including definitions, will control.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

The term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components. In addition, as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of "consisting of " and/or "consisting essentially of".

As used in the specification and claims, the term "and/or" used in a phrase such as "A and/or B" herein is intended to include "A and B", "A or B", "A", and "B" . As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As used herein, the term "virustatic" refers to inhibition of the growth and/or development and/or the replication of viruses, which is different from destruction of viruses. Typically, the inhibition effect is obtained by coating of virus capsids or blocking ceil surface receptors effectively, thereby creating a barrier to interaction between a virus and a cell. However, a virus remains active, can be released and can further infect cells. As used herein, the term "virucidal" refers to neutralization and/or destructions of a virus.

Interaction with virucidal compounds alters the virus, rendering it inert, and thereby prevents further infections.

As used herein, the term "biocompatible" refers to compatible with living cells, tissues, organs, or systems, and having no risk of injury, toxicity, or rejection by the immune system.

The term "alkyl" used alone or in combination with other groups should be understood to include straight chain and branched hydrocarbon groups having from 4 to 50, preferably 6 to 20 carbon atoms. Alkyl groups may be optionally substituted with one or more substituents. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n- propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, n-hexyl .

A biomimetic strategy has been developed to develop broad-spectrum virucidal drags. To limit toxicity, it has been decided to stay away from known bio-toxic approaches and to concentrate on mimicking cell-receptor, so to strongly attach to their corresponding viral ligand and generate local viral deformation that would ultimately lead to irreversible viral mutations, possibly to viral disassembly. To achieve broad-spectrum efficacy, it was aimed at virus-cell interactions that are common to many viruses. One of these interactions is that between viruses and cell-surface attachment receptors that represent the very first step of the virus replicative cycle. Many viruses, including HIV-1 , HSV, HCMV, HPV, Respiratoiy syncytial virus (RSV) and filovirases, exploit heparan sulfate proteoglycans (HSPGs) as attachment receptors, as HSPGs are expressed on the surface of almost all eukaryotic cell types. The binding between viruses and HSPGs usually occurs via the interaction of stretches of basic amino acids on viral proteins (basic domains) with the negatively charged sulfated groups of heparan sulfate (HS) chains in the glycocalix of the cell surface.

In the present invention, a series of nanoparticles with a very high density of long sulfonic acid terminated molecules have been designed, in order to induce their strong multivalent binding leading to irreversible changes in HSPG dependent viruses either enveloped (e.g. HSV, RSV, Lentivims, Dengue virus) or naked (e.g. HPV). An aspect of the invention provides a virucidal metallic nanoparticle comprising multiple (several) alkyl sulfonate ligands, that provide the attachement receptor for viruses, such as HSPG binding viruses and optionally multiple (several) additional alkyl ligands. In preferred embodiments of the virucidal metallic nanoparticles of the invention, the alkyl sulfonate ligand (group) is -Z-CH₂-(CH₂)y-SO₃-, wherein

Z is absent or selected from the group comprising O, S, and -C(=0)NH-(CH₂)2-X, y is at least 5, preferably y is 5 to 20, preferably y is 7 to 1 1 or 5 to 1 1 , most preferably y is

5, 10 or 11. In other embodiments, y is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10. In other embodiments, y is at maximum 100, at maximum 70, at maximum 50, at maximum 25, at maximum 20, at maximum 15.

X is substituted or unsubstituted cyclo-C₅-C₇-alkyl or aryl, wherein one or more substituents are selected from the group comprising Ci -CVa!kyl and -OH. Preferably X is aryl substituted with two -OH.

In other preferred embodiments, the alkyl sulfonate ligand is 1 1 -mercapto-l - undecanesulfonate (MUS) or 6-((3,4-dihydroxyphenethyl)amino)-6-oxohexane- 1 -sulfonate

(DOS). In preferred embodiments of the virucidal metallic nanoparticles of the invention, metallic nanoparticles are gold nanoparticles or iron oxide nanoparticles.

In other preferred embodiments of the virucidal metallic nanoparticles of the invention, the virucidal metallic nanoparticles of the invention comprise several (multiple) additional alkyl ligands. In a preferred embodiment, the additional alkyl ligand is -Q-CH₂-(CH₂)w, wherein Q is O or S or absent and w is at least 4, preferably w is 4 to 20, preferably w is 7 to 1 1 , most preferably w is 10 or 1 1. In other embodiments, w is ar least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 . In other embodiments, w is at maximum 100, at maximum 70, at maximum 50, at maximum 25, at maximum 20, at maximum 15. In other preferred embodiments, the additional alkyl ligand is octalen thiol (OT).

In some embodiments of the virucidal metallic nanoparticles of the invention, the ratio between alkyl sulfonate ligands and alkyl ligands is about 2: 1 , from 1.5: 1 to 2.5: 1, from 1.5:0.5 to 2.5: 1.5. in further embodiments, nanoparticles were designed with the comparable ligand shell but a more biocompatible core than gold. Preliminary results with 6-((3,4- dihydroxyphenethyl)amino)-6-oxohexane-l -sulfonate (DOS) coated iron oxide NPs show virucidal activity similar to their gold counterpart, see Fig. 22, indicating the importance of the particles molecular coating rather than their core material.

In the context of the present disclosure, "multiple alkyl sulfonate ligands" and "multiple alkyl ligands" refers to the virucidal nanoparlicle core that is coated, partially or completely, by multiple (several) alkyl sulfonate ligands of the invention and optionally by multiple (several) alkyl ligands of the invention. The coating can be homogenous, unstmctured or structured. In some embodiments, the virucidal metallic nanoparticle comprises very high density of alkyl sulfonate ligands of the invention. In some embodiments, the virucidal metallic nanoparticle comprises several (at least six) alkyl sulfonate ligands of the invention in close proximity. In other embodiments, the virucidal nanoparticles are coated with the multiple alkyl sulfonate ligands of the invention that can be the same or different and with the optional alkyl ligands of the invention that can be the same or different.

Some examples of nanoparticles of the invention are provided in Table 1.

Table 1 : examples of nanoparticles of the invention.

Contrary to the general knowledge, it was unexpectedly found that the specific lengh of the alkyl sulfonate ligands, i.e. 5 to 20 carbons, preferably 5 to 11, provide not only attachement receptor for HSPG binding viruses, but also provide virucidal effect, which is different from the known virustatic effect.

The virucidal metallic nanoparticles of the invention provide virucidal activity at low concentrations, such as at micro molar levels and/or nano molar levels, against a wide range of viruses, such as herpes simplex virus (HSV), human papillomavirus virus (HPV), respiratory syncytial virus (RSV), dengue virus and lentivirus (a human immunodeficiency virus (HIV) derived virus).

To evaluate the inhibitory activity of nanoparticles of the invention, the following viruses were used: Herpes simplex type 1 (HSV-1), Herpes simplex type 2 (HSV-2), Human Papilloma virus- 16 pseudoviruses (HPV- 16), Respiratory syncytial virus (RSV), VSV pseudo-typed lentivirus (LV-VSV-G), and Dengue virus. All of the viruses above are HSPG dependent viruses. Adenovims-5 (ADS), a non-HSPG dependent virus, was used as a control. To mimic HSPG, nanoparticles coated with 1 1 -inercaptoundecansulphonate (MUS) were prepared, as this ligand has a long hydrophobic backbone terminating with a sulfonic acid, allowing its terminal group to move with some freedom. Consequently, particles coated with MUS are ideal for multivalent binding, in this case the binding of multiple sulfonic acids to the HSPG-interacting motifs on the virus surface. Gold nanoparticles coated with MUS ligands were selected, as they are the simplest non-toxic particles that can be synthesized with these ligands. Other particles selected in the present invention are the particles coated with a 2: 1 mixture of MUS and 1 -octanethiol (OT), as they are the least toxic, most soluble and protein resistant version of MUS-coated gold particles that have been studied. All nanoparticles used (either as drugs or as controls) are summarized in Table 2, and all synthetic methods and characterization are presented in the Example section.

Table 2. Overview of the tested nanoparticles

Determined from TEM images and expressed as average diameter ± standard deviation.

:Ligand ratio calculated from Ή NMR analysis after decomposition of the core, see Figure 5. Viral Inhibition Results

All viruses were pre-incubated with different doses of gold nanoparticles (Au-NPs) for 1 h at 37°C and 5% CO₂, then the mixture was added to the cell culture (see Example Section for virus- specific protocol details, initial viral load, and cell types), and infectivity was tested 24-72 h post infection. For the GFP expressing viruses (LV-VSV-G, AD-5 and HPV-16) the infectivity was quantified by flow cytometry, while plaque assays were used for wild-type viruses. Table 3 summarizes the results. It is noteworthy, that the MUS containing particles (i) are indeed nontoxic at these concentrations showing favourable selectivity indexes, (ii) are able to inhibit infection only for HSPG dependent viruses (no inhibition is observed for AD5), and that (iii) all ECso are in the nanomolar range (with respect to moles of nanoparticles, see Example Section for calculations). It is important to underline that the use of the monomeric sulfonated ligand (MUS) without NPs was found not to be effective in inhibiting LV-VSV-G. One possible explanation for the lack of inhibition for MUS can be interactions between various chemical groups on the surface of viruses and the thiols at the end of the ligands. To make sure that these interactions were not a major effect, it was also tested (against all viruses) the efficacy of sodium undec- 10- enesulfonate (pre-MUS), a molecule equivalent to MUS but lacking the thiol end-group. No inhibitory activity was found. Altogether these results prove the importance of the multivalent interaction in inhibiting the viruses at appreciable concentrations.

Table 3 : Inhibitory activity of nanoparticles

To further test that the MPs affect infcctivity by mimicking the attachment receptor for HSPG- binding virases, a series of control experiments were performed. Au-NPs coated with 11- mercaptoundecylphosphoric acid (MUP) Iigands (Fig. 8) were synthesized, thus creating particles of similar size, ligand and charge density to the MUS-NPs but replacing the sulfonate with phosphonate groups. In contrast to the MUS-NPs, the MUP-NPs showed no inhibitory activity when mixed with pseudo-lentivirus (LV-VSV-G), highlighting the importance of the sulfonic acid group for the activity of the particles. The inhibitory activity of 15 nm in diameter citrate coated Au-NPs was tested and found none.

Additionally, a control experiment was conducted using adeno-associated virus (AAV) in which two serotypes (AAV2 and AAV5) have different cell surface attachment receptors. AAV2 recognizes host cells through HSPGs while AAV5 binds to sialic acid as its primary cell attachment receptor; CHO-Kl cells are permissive to both serotypes. As expected, incubation of these virases with MUS:OT-NPs, showed inhibition only for the AAV2 serotype, indicating selectivity over the attachment ligand (Fig. 11 ). Virucidal Results

The nanoparticles of the invention were designed by using many long (and hence reasonably flexible) ligands that cooperatively bind to the virus, to achieve a strong multivalent binding at long distances, and consequently irreversible local deformations. First, the ability of the nanoparticles of the invention to inhibit viral attachment was verified, as is known for heparin (Fig. 12). Then, they were tested for irreversible inhibitory activity through virucidal assays, to verify that MUS coated NPs can irreversibly inhibit viruses. The latter assay consists of an incubation of the virus and drags at a concentration corresponding to the EC90 for a given amount of time and the subsequent evaluation of the residual infectivity of the virus through serial dilutions of the inoculum. If the effect is solely virastatic, the viral infectivity is fully recovered upon dilution, as shown herein for heparin against HSV-2 (Fig. 1A, top). Additionally, MES coated Au-NPs of the same type presented in literature were synthetized and tested them against HSV-2. It was found these particles to have inhibitory activity in the nanomolar range, but virucidal tests showed full recovery of the viral infectivity indicating a simple virustatic inhibitory mechanism (Fig. 1A, middle) similar to the one found for heparin. If irreversible changes are induced in the viruses, the infectivity is never regained at all dilutions tested, even though the dilution leads to a final concentration lower than the active dose. MUS:OT-NPs also showed nanomolar inhibition of HSV-2 infectivity but, in contrast with to heparin and MES- NPs, no infectivity was regained upon dilution (Fig. 1A, bottom), indicating a strong irreversible effect (virucidal). The difference between MES and MUS coated nanoparticles clearly indicates the importance of the long NP ligands, needed to achieve long-range strong multivalent interactions. All HSPG-binding viruses showed irreversible loss of infectivity when incubated with MUS:OT-NPs, although to a different extent (Fig. IB). The HSV-2 virucidal tests were performed also at different time points, as shown in Fig. IC. While the virastatic effect is immediate (as shown by dose response curve at time Oh in Fig. 13), the virucidal activity develops over time, with the effect being almost complete after 30 min. Indeed, when viruses and nanoparticles were mixed and immediately added to cells, the inhibitory potency is reduced as compared to the pre-incubation experiment, confirming the time-dependent virucidal effect (Fig. 13).

Investigations of the Irreversible Changes in the Virus

To further elucidate the fate of the viruses, transmission electron microscopy (TEM) was performed on HSV-2 viruses exposed to MUS:OT-NPs. HSV-2 is an enveloped vims, consequently, its interaction was studied primarily using cryo-TEM, as this procedure is known not to affect the envelope itself (this is at least partially confirmed by the three-dimensional reconstruction of the images, see discussion below). Observations were confirmed also by using dry, negatively stained TEM imaging, finding no major differences. Fig. 2 shows cryo-TEM (panel A, top) and dry negative stain (panel A, bottom) images of viruses with and without NPs. In both imaging techniques, NPs can be clearly observed close to the viral capsids, and in both cases viruses appear to have lost their integrity upon exposure to the nanoparticles. TEM is a two-dimensional projection of a three-dimensional image so it is hard to estimate the true distance between the capsid and the particles. In a few cases, imaging the samples at various angles was successful so that a three-dimensional tilt series of the images could be made. In these cases it is clear that the particles are actually a few nanometers from the virus capsids, in good agreement with the expected thickness of the envelope. Interestingly, clusters of particles at specific locations close to (but not exactly at) the viral capsid were observed; these clustering of particles was interpreted as the location on the envelope of the HSPG ligand (see arrows in the image shown in the white box in Fig. 2B, bottom). The three-dimensional reconstruction of the images confirms this interpretation.

The results of imaging investigations are that (1) incubating NPs with virus one can observe that almost all viruses become covered with nanoparticles. (2) In cryo-TEM, it is possible to observe a sizeable fraction of viruses that are fully covered with NPs, which appear to have lost integrity (Fig. 2B, bars at 90', and Fig. 14). Similarly, in negative stain images, a fraction of viruses can be observed with odd shapes (Fig. 2A, bottom). These viruses appear to be more deformed than the ones observed in cryo-TEM, but in this case, harsh drying conditions could be at least partly responsible for their deformation. (3) As shown in Fig 2B, images taken at different time points in cryo-TEM, reveal that viruses get increasingly covered with NPs over time. At the endpoint (90 minutes), the majority of the viruses are either associated with clusters or fully covered with NPs. It is believed that the progression of the number of particles per virus with time eventually leads to the viral rupture (i.e. irreversible change). The particles -upon multivalent binding to the HSPG ligands on the virus- deform locally the envelope, approaching the capsid. When particles are bound to many attachment ligands the envelope is disrupted allowing for unspecific binding of the nanoparticles directly to the capsid. Similar effects of nanoparticle clusters attached to viruses, or completely covered viruses were observed also for HPV-16 pseudovirions (PsVs) (Fig. 15). As a control, HSV-2 viruses incubated with non-viracidal NPs i.e. gold nanoparticles coated with 6-mercaptohexyldiethyleneg!ycol (see Table 1 and 2, EG2-OH) were also imaged as none sulfonic acid-containing particles. Their association with HSV-2, as studied in TEM, was sporadic and it was never possible to observe more than a few NPs associated to the virus, as shown in Figure 16.

Mechanistic Understanding via Simulations

To test whether the virucidal activity changed the physico-chemical properties of the whole population of viruses (as suggested by the TEM studies), it was reverted to studies of gel electrophoresis on LV-VSV-G (as for this pseudo-virus it is simple to achieve the high titers necessary to have reliable data for this type of analysis). Figure 17 shows that the virus, either stained for protein or for DNA, runs differently upon interaction with MUS:OT-NPs compared with the virus alone. A shift and a spread of the viral bands in presence of the nanoparticles are observed, suggesting a possible degradation of the viruses and therefore a smaller and heterogeneous size of the fragments after running on the gel. This result indicates a substantial change in the properties of the whole population of viruses.

In order to understand how MUS-NPs can induce irreversible changes in the capsids of HSPG- specific viruses, it was performed atomistic molecular dynamics (MD) simulations of such NPs interacting with the capsid of HPV-16 and other viruses (see Example Section, and Figs. 18-20 for details). Figure 3 shows two examples of systems examined in physiological solutions, where NPs placed at the virus surface interact with the HPV-16 capsid L1 proteins. In Figure 3 A, a small MUS-NP (2.4 ran core) binds to a single L1 protein pentamer via HSPG-specific binding sites, while in Fig. 3B, a large MUS NP (5 nm core) binds to two L1 pentamers in their native configurations. Except for their mutual binding, the proteins were not fixed to other structures, in contrast to real viruses. The modelled systems freely evolved from their initial states, in which NPs were positioned close to the solvent-exposed I 1SPG binding sites (amino acid residues K278, K356, K361, K54 and K59).

Figure 3 demonstrates that highly selective, strong, and long-range multivalent binding of the negative sulfonate groups of MUS-NPs to positive HSPG-binding lysine residues can induce large stress and subsequent deformations in L1 capsid protein complexes. Figure 3A shows that a small NP binds tightly to an L1 pentamer. In 25 ns simulations, four stable interaction points form between NP terminal sulfonate groups and L1 HSPG-binding sites, while the surface of the L1 pentamer becomes gradually deformed. Figure 3B shows that when a larger NP binds to two neighbouring L1 pentamers (in the arrangement observed within the complete HPV capsid), the number of binding sites gradually increases in time, which induces mutual reorientation of the unsupported L1 pentamers. In the complete viral capsid, the reorientation of L1 proteins is likely to be slow and occurring opportunistically during natural fluctuations of the capsid, because of their stable array-like supported arrangement. Despite their slow but steady progression, the local changes could accumulate and ultimately lead to larger irreversible changes in the virus capsid surface.

The results of MD simulations, shown in Fig. 3, can be used to estimate effective forces that NPs exert on L1 capsid proteins, thus providing a better insight into the capsid deformation processes. In a binding configuration, a sulfonate group binds to positively charged amine groups of lysine residue with a relatively large Gibbs free energy of

6 kcal/mol. In MUS-NP the nature of the ligands is consistent with binding events at different sites that are coordinated and so their effects are coherently added. Therefore, as shown in Fig. 3C, by considering the increase of binding energy (sites) per a typical NPs motion on the capsid surface, one can evaluate an effective force that drives the process forward, In Fig. 3A, the NP increases the

number of interaction points from 1 to 4, while moving by This gives an effective

force of F ~ 125 pN.

Another aspect of the invention discloses a pharmaceutical composition comprising an effective amount of one or more virucidal metallic nanoparticles of the invention and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

As to the appropriate excipients, carriers and diluents, reference may be made to the standard literature describing these, e.g. to chapter 25.2 of Vol. 5 of "Comprehensive Medicinal Chemistry", Pergamon Press 1990, and to "Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete", by H.P. Fiedler, Editio Cantor, 2002. The term "pharmaceutically acceptable carrier, excipient and/or diluent" means a carrier, excipient or diluent that is useful in preparing a pharmaceutical composition that is generally safe, and possesses acceptable toxicities. Acceptable carriers, excipients or diluents include those that are acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable carrier, excipient and/or diluent" as used in the specification and claims includes both one and more than one such carrier, excipient and/or diluent.

Optionally, the pharmaceutical composition of the present invention further comprises one or more additional active agents, preferably anti-viral agents.

The virucidal metallic nanoparticle compounds of the invention that are used in the methods of the present invention can be incorporated into a variety of formulations and medicaments for therapeutic administration. More particularly, a compound as provided herein can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers, excipients and/or diluents, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracranial and/or intratracheal administration. Moreover, the compound can be administered in a local rather than systemic manner, in a depot or sustained release formulation. The compounds can be formulated with common excipients, diluents or carriers, and compressed into tablets, or formulated as elixirs or solutions for convenient oral administration, or administered by the intramuscular or intravenous routes. The compounds can be administered transdermal ly, and can be formulated as sustained release dosage forms and the like. The compounds can be administered alone, in combination with each other, or they can be used in combination with other known compounds.

Suitable formulations for use in the present invention are found in Remington's

Pharmaceutical Sciences (Mack Publishing Company (1985) Philadelphia, PA, 17th ed.), which is incorporated herein by reference. Moreover, for a brief review of methods for drug delivery, see, Langer, Science (1990) 249: 1527-1533, which is incorporated herein by reference.

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi permeable matrices of solid hydrophobic polymers containing the compound of the invention, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methaerylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and [gamma] ethyi- L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT(TM) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyric acid. The virucidal metallic nanoparticle compounds of the present invention may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymcthylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The pharmaceutical compositions described herein can be manufactured in a manner that is known to those of skill in the art, i.e., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The following methods and excipients are merely exemplary and are in no way limiting. For injection, the compound (and optionally another active agent) can be formulated into preparations by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. Preferably, the compounds of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Preferably, pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The amount of a virucidal metallic nanoparticle compound of the invention that can be combined with a carrier material to produce a single dosage form will vary depending upon the viral disease treated, the mammalian species, and the particular mode of administration. It will be also understood, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular viral disease undergoing therapy, as is well understood by those of skill in the area.

Further aspect of the invention provides a method of treating and/or preventing viral infections and/or diseases associated with viruses, comprising administering to a subject in need thereof, a therapeutically effective amount of the virucidal metallic nanoparticles of the invention.

Another aspect of the invention provides the virucidal metallic nanoparticles of the invention for use in treating and/or preventing viral infections and/or diseases associated with viruses.

In order to develop an effective antiviral strategy the used drags have to act mainly after infection. MUS.OT-NPs were effective also after virus infection of cells. Cells were infected with wild-type HSV-2 (multiplicity of infection, MOI 0.01 plu/^'cell) for 2 hours at 37°C. After the removal of the viral inoculum, different doses of MUS:OT-NPs were added to the cell monolayers. Cells and supernatants were harvested when the untreated wells exhibited a cytopathic effect of the whole monolayer. The cell free supernatants were then titrated. It was determined that MUS:OT-NPs had an EC50 of 4.4 μg/mL, with complete inhibition at 400 Hg/mL and 3 logs reduction at 80 μg/mL (Fig. 21). Thus the NPs can either prevent infection or block an ongoing infectious process depending on whether they inactivate the viras inoculum or the viral progeny.

In further embodiments, nanoparticles were designed with the comparable ligand shell but a more biocompatible core than gold. Preliminary results with 6-((3,4- dihydroxyphenethyl)amino)-6-oxohexane- 1 -sulfonate (DOS) coated iron oxide NPs show virucidal activity similar to their gold counterpart, Fig. 22, indicating the importance of the particles molecular coating rather than their core material.

The nanoparticles of the invention provide medically relevant virucidal drugs to fight viral infections. The results found so far show outstanding virucidal activity over HSV-2 and LS-

VSV-G, and good activity versus HPV and RSV. It should be stressed that the prevention and treatement strategy proposed herein is intrinsically broadband, allowing the potential prevention and treatment of multiple infections with a single ding, a great advantage mostly in virology where rapid and at times unexpected mutations occur. For example, Zika virus has emerged as a great threat, whilst West Nile, Yellow Fever, and Dengue are an ever growing threat. All these viruses belong to the Flaviridae family, and are HSPG-binding viruses. Preliminary results with MUS particles show nanomolar virucidal efficacy over Dengue 2 (see Fig. IB and Fig. 23). Similarly, the Filoviridae family contains several human pathogens causing haemorrhagic fevers, including Ebola virus, for which drugs are urgently needed. All of them bind HSPGs as attachment receptors, and thus are potentially susceptible to the antiviral NPs presented herein. Overall, the technological innovation presented herein provides a breakthrough for the development of treatments for many worldwide threatening viral infections.

In some embodiments, the viruses are HSPG binding viruses. In other embodiments, the viruses are selected from, but not limited to, the group comprising herpes simplex virus

(HSV), human papillomavirus virus (HPV), respiratory syncytial virus (RSV), dengue virus and lentivirus (a human immunodeficiency virus (HIV) derived virus).

As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject beining infected by a virus, such as HSPG binding viruses. However, in other embodiments, the subject can be a healthy subject or a subject who has already undergone a treatment. The term does not denote a particular age or sex. Thus, adult, children and newborn subjects, whether male or female, are intended to be covered.

"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already being infected by a virus, such as HSPG binding vims, as well as those in which the viral infection is to be prevented. Hence, the mammal, preferably human, to be treated herein may have been diagnosed as being infected by a virus, such as HSPG binding virus, or may be predisposed or susceptible to be infected by a virus, such as HSPG binding virus. Treatment includes ameliorating at least one symptom of, curing and/or preventing the development of a disease or condition due to viral infection. Preventing is meant attenuating or reducing the ability of a virus to cause infection or disease, for example by affecting a post-entry viral event. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals or pet animals, such as dogs, horses, cats, cows, monkeys etc. Preferably, the mammal is human.

The term "therapeutically effective amount" refers to an amount of the virucidal compound of the invention effective to alter a virus, such as HSPG binding virus, and to render it inert, in a recipient subject, and/or if its presence results in a detectable change in the physiology of a recipient subjet, for example ameliorates at least one symptom associated with a viral infection, prevents or reduces the rate transmission of at least one viral agent. Another aspect of the invention provides a virucidal composition comprising an effective amount of the virucidal metallic nanoparticle compound of the invention and optionally at least one suitable carrier. "An effective amount" refers to the amount sufficient for altering viruses, and/or destroying viruses and/or neutralizing viruses; i.e. sufficient for obtaining virucidal effect. In an embodiment, the suitable carrier is selected from the group comprising stabilisers, fragrance, colorants, emulsifiers, thickeners, wetting agents, or mixtures thereof. In another embodiment, the virucidal composition can be in the form of a liquid, a gel, a foam, a spray or an emulsion. In a further embodiment, the virucidal composition can be an air freshener, a sterilizing solution or a disinfecting solution. Another aspect of the invention provides a device (or a product) comprising the virucidal composition of the invention and means for applying and/or dispensing the virucidal composition. In another embodiment, the means comprise a dispenser, a spray applicator or a solid support soaked with the virucidal composition. In another embodiment, the support is a woven or non- woven fabric, a textile, a paper towel, cotton wool, an absorbent polymer sheet, or a sponge.

Another aspect of the invention provides a method of disinfection and/or sterilization using the virucidal metallic nanoparticles of the invention or the virucidal composition of the invention.

In a preferred embodiment, the method of disinfection and/or sterilization comprises the steps of (i) providing at least one virucidal metallic nanoparticle of the invention or the virucidal composition of the invention, (ii) contacting a virus contaminated surface or a surface suspected to be contaminated by viruses with the at least one virucidal metallic nanoparticle of the invention or the virucidal composition of the invention for a time sufficient to obtain virucidal effet. Preferably the virus is HSPG binding virus; more preferably virus is selected from the group comprising herpes simplex virus (HSV), human papillomavirus virus (HPV), respiratory syncytial virus (RSV), dengue virus and lentivirus (a human immunodeficiency virus (HIV) derived virus). In some embodiments, the virus contaminated surface is human or animal skin. In other embodiments, the virus contaminated surface is a non-living surface, such as medical equipments, clothing, masks, furnitures, rooms, etc.

Another aspect of the invention provides a use of the virucidal metallic nanoparticles of the invention or the virucidal composition of the invention for sterilization and/or for disinfection. In some embodiments, sterilization and disinfection is for virus contamined surfaces or surfaces suspected to be contaminated by viruses. Preferably the virus is HSPG binding virus; more preferably virus is selected from the group comprising herpes simplex virus (HSV), human papillomavirus virus (HPV), respiratory syncytial virus (RSV), dengue virus and lentivirus (a human immunodeficiency virus (HIV) derived virus). In some preferred embodiments, the surfaces are human or animal skin. In other preferred embodiments, the surfaces are non-living surfaces, such as medical equipments, clothing, masks, furnitures, rooms, etc. In an embodiment, the virucidal composition is used as virucidal hand disinfectant for frequent use. In another embodiment, the virucidal composition is applied by spraying. In a further embodiment, the virucidal composition is applied on a protective mask. Further instances of the present disclosure:

1. A virucidal metallic nanoparticle comprising multiple alkyl sulfonate ligands that provide the attachement receptor for viruses, and optionally multiple additional alkyl ligands, wherein

the alkyl sulfonate ligand is , and wherein

Z is absent or selected from the group comprising O, S, and

y is 5 to 20,

X is substituted or unsubstituted cyclo-C₅-C₇-alkyl or aryl, wherein one or more substituents are selected from the group comprising C₁-C₄-alkyl and -OH. and the optional additional alkyl ligand is

, and wherein

Q is absent or selected from O or S,

w is 4 to 20.

2. The virucidal metallic nanoparticle of instance 1 , wherein y is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10.

3. The virucidal metallic nanoparticle of instance 1 or 2, wherem y is at maximum 100, at maximum 70, at maximum 50, at maximum 25, at maximum 20, at maximum 15.

4. The virucidal metallic nanoparticle of any one of instances 1 to 3, wherein w is at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1. 5. The virucidal metallic nanoparticle of any one of instances 1 to 3, wherein w is at maximum 100, at maximum 70, at maximum 50, at maximum 25, at maximum 20, at maximum 15.

6. The virucidal metallic nanoparticle of any one of instances 1 to 5, wherein

wherein X is aryl substituted with two -OH,

y is 5 to 1 1 7. The virucidal metallic nanoparticle of any one of instances 1 to 6, wherein the alkyl sulfonate ligand is 1 1 -mercapto- 1 -undecanesulfonate (MUS) or 6-((3,4- dihydroxyphenethyl)amino)-6-oxohexane- l -sulfonate (DOS).

8. The virucidal metallic nanoparticle of any one of instances 1 to 7, wherein the additional alkyl ligand is octalen thiol (OT).

9. The virucidal metallic nanoparticle of any one of instances 1 to 8, wherein the metallic nanoparticle is gold nanoparticle or iron oxide nanoparticle.

10. The virucidal metallic nanoparticle of any one of instances 1 to 9, wherein the ratio between alkyl sulfonate ligands and alkyl ligands is from 1 .5: 1 to 2.5: 1.

1 1. The virucidal metallic nanoparticle of any one of instances 1 to 10, wherein the ratio between alkyl sulfonate ligands and alkyl ligands is about 2: 1.

12. The virucidal metallic nanoparticle of any one of instances 1 to 1 1 , wherein the alkyl sulfonate ligands are the same or different.

13. The virucidal metallic nanoparticle of any one of instances 1 to 12, wherein the alkyl ligands are the same or different.

14. A pharmaceutical composition comprising an effective amount of one or more virucidal metallic nanoparticles of any one of instances 1 to 13 and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

15. The virucidal metallic nanoparticle of any one of instances 1 to 13 for use in treating and/or preventing viral infections and/or diseases associated with viruses.

16. The virucidal metallic nanoparticle for use in treating and/or preventing viral infections and/or diseases associated with viruses of instance 15, wherein the viruses are

HSPG binding viruses. 17. The virucidal metallic nanoparticle for use in treating and/or preventing viral infections and/or diseases associated with viruses of instance 16, wherein the viruses are selected from the group comprising herpes simplex virus (HSV), human papillomavirus virus (HPV), respiratory syncytial virus (RSV), dengue virus and lentivirus (a human immunodeficiency virus (HIV) derived virus),

1 8. A virucidal composition comprising an effective amount of the virucidal metallic nanoparticles of any one of instances 1 to 13 and optionally at least one suitable carrier. 19. A method of disinfection and/or sterilization comprising using the virucidal composition of instance 18 or the virucidal metallic nanoparticles of any one of instances 1 to 13.

20. A device comprising the virucidal composition of instance 18 or the virucidal metallic nanoparticles of any one of instances 1 to 1 3 and means for applying or dispensing the virucidal composition or the virucidal metallic nanoparticles.

21 . A use of the virucidal metallic nanoparticles of any one of instances 1 to 13 or the virucidal composition of the instance 18 for sterilization and/or for disinfection.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the scope of the invention. Examples

Materials and Methods

All chemicals were purchased from Sigma Aldrich and used as is unless otherwise specified. All solvents purchased were reagent grade and purged with nitrogen gas for more than 30 min prior to the reaction.

Synthesis of 11-mercapto-1-undecanesulfonate (MUS) ligand:

MUS ligands were synthesized according to a modified literature method (Vema, A. et al, 2008). A gram-scale synthesis is outlined, adaptable to scales from 1 to 40 g of precursor. A synthesis using 25 mL of the precursor is described.

Sodium undec- 10-enefulfonate: 1 1 -bromo- 1 -undecene (25 mL, 11 1.975 mmol), Sodium Sulfite Na₂SO₃ (28.75 g, 227.92 mmol) benzyltriethyl-ammonium bromide ( 10 mg) were added to a mixture of 200 mL methanol and 450 mL Dl-water (4:9 McOH:H₂O ratio) in a 1 L round bottom flask. The mixture was refluxed at 102°C for 48h. The mixture was extracted with diethyl ether 5 times, (5 x 400 ml), and the aqueous phase was evaporated in a rotary evaporator. The white powder was dried under high vacuum, suspended in pure ethanol and filtered. The methanolic solution was evaporated, and the process was repeated twice, to decrease the amount of inorganic salts. Usually, about 33 g of white, methanol soluble power was collected at this scale. Ή-NMR (D20); 5.76 (m, 1H), 4.78 (m, 2H), 2.69 (t, 2H), 1.53 (m, 2H), 1.11 (br s, 12H).

Sodium l l-acetylthio-undecanefulfonate: Sodium undec- 10-enefulfonate (33 g, 147.807 mmol) was dissolved in 500 ml of methanol. A 2.6 times excess of thioacetic acid (27.324 mL, 384.3 mmol) was added to the solution and stirred in front of a UV lamp overnight ( 12h). The solution was evaporated in a rotary evaporator until the solid residue turned orange-red. The solid was washed with diethyl ether, until no colored material could be removed. The solid was dried under high vacuum, and then dissolved in methanol producing a yellow solution. About 3 g of carbon black was added to the solution, vigorously mixed, and the mixture was filtered through celite in a fluted filter paper. The filtered solution was clear, the solvent completely evaporated and about 35 g of white solid was collected. 'H-NMR (D20): 2.69 (t, 4H), 2.17 (s, 3H), 1.53 (m, 2H), 1.39 (m, 2H), 1.1 1 (br s, 1411).

1 1 -mercapto- 1 -undecanesulfonate (MUS): Sodium 1 1 -acetylthio-undecanefulfonate was refluxed at 102°C in 400 mL of IM HCl for 12 h. 200mL of IM NaOH was added to the final solution, additional 400 niL of Dl-water was added to create a 1 L volume. The clear solution was kept at 4°C and crystallized overnight. The viscous white product was centrifuged down in 50 mL falcon tubes, and dried under high vacuum. 12 g (about 30% yield) of methanol soluble MUS is collected from this purification step. More material can be extracted from the supernatant of the centrifugation step, by reducing volume and keeping it at 4°C. 'H-NMR (D20): 2.69 (t, 4H), 2.34 (t, 311), 1.53 (m, 2H), 1.39 (m, 211), 1.1 1 (br s, 14H). Mass spectrum (ESI) found m/z = 291.107, calculated mass 290.42 g/mol.

Synthesis of all MUS and MUS:OT gold nanoparticles:

1.2 mmol of gold salt (HAuCU) was dissolved in 200 mL of ethanol and 1.2 mmol of the desired thiol ligand mixture (two MUS to each OT, for MUSiOT NPs) was added while stirring the reaction solution, then a saturated ethanol solution of sodium borohydride (NaBH₄) was added drop-wise over 2 h. The solution was stirred for 3 h and the reaction flask was then placed in a refrigerator overnight. The product was washed several times (5 to 10) by suspending and centrifuging (5500 rpm) it in methanol, ethanol and then acetone. Finally, the product was washed 5 times with Dl-water using Amicon® Ultra- 15 centrifugal filter devices (30k NMWL). The particles were then suspended in a minimal amount of water and freeze-dried to yield ~ 250 mg of a black powder. After etching of MUS:OT-NPs (lOmg) in a solution of 15 mg Iodine (Acros) in 0.6 mL of MeOD-d₄ (Sigma) for 30 min under sonication, the spectra revealed a 34% OT content, i.e. a 2: 1 MUS:OT stoichiometry, Figure 5.

Synthesis of Oligo-Ethylene Glycols gold nanoparticles, EG₂-OH NPs:

EG₂-OH NPs were synthesized according to a modified Stucky procedure. The reaction was performed in a 100 ml 3-neck round bottom flask in a total volume of 20 ml of a mixture of dimethylformamide (DMF) and 20% Methanol, at 100 °C in an oil bath under reflux. Briefly, 0.25 mmol of chloro(tripheny!phosphine)gold(I) and 0.125 mmol of EG?-OH, i.e. HS-(CH₂)₆)- (ProChimia Surfaces, Poland) were dissolved in 15 ml of a mixture of

dimethylformamide (DMF) and 20% Methanol and then stirred for at least 20 min. When the reaction temperature is reached, 2.5 mmol of borane tert-butylmine complex previously dissolved in 5 ml of solvent mixture were added in one portion to the reaction mixture. The solution colour turns from transparent to light brown and then slowly to deep red. The reaction is left stirring at 100 °C for 1 h 30 min. Afterward, the heating was turned off and the solution left stirring for additional 3 h. The nanoparticles were precipitated with acetone and kept overnight at 4°C. A black precipitate of Au-NPs was suspended and centrifuged at 3220 g for 20 min in acetone for five times. The black precipitate was left to dry under vacuum. Subsequently the black precipitate was dissolved in ultrapure deionized water (18.2 ΜΩ cm at 25 °C) and dialysed extensively against ultrapure deionized water. Removal of residual salts from the synthesis was checked by measuring the conductivity of a 0.5 mg/ml solution of nanoparticles, which had to be below 5 LiS/cm. The solution obtained after the dialysis was then concentrated through diafiltration with Vivaspin 6 ml to a final volume between 1-2 ml. This volume was then freeze dried and lyophilized and the powder recovered. The NPs were imaged by TEM, the average core diameter was 6.2 ± 0.8 nm, Figure 7.

Synthesis of MUP gold nanoparticles, MUP-NPs:

Separately 12-mercaptoundecylphosphoric acid (MUP) (255 mg, 0.9 mmol) was dissolved in ethanol (20 ml, Fluka, Puriss > 99.8%) and NaBH₄ (2 g) was dissolved in ethanol (200 ml, Fluka). Both solutions were then sonicated to aid dissolution and filtered to remove any insoluble residue. In a third container gold (III) chloride trihydrate (354 mg, 0.9 mmol) was dissolved in ethanol (200 ml, Fluka). The ligand (MUP) solution was then added to the gold salt solution with stirring for 10 minutes. To this vigorously stirred solution was then added dropwise the NaBI U solution. After complete addition of the reducing agent the mixture was stirred for l h and then the reaction flask was stored overnight at 4°C to precipitate the nanoparticles. The nanoparticles were then spun down (5000 rpm). The supernatant was removed and the nanoparticles re- dispersed in 45 ml of ethanol. Ethanol washing was repeated 3 times. The residue was then dispersed in water (15 ml) and filtered through Amicon® Ultra-50 centrifugal filter devices (30k MW cut off) to further wash the particles. This was repeated extensively until the water removed no longer foamed when shaken. The nanoparticles were then dialyzed (8k MW cut off) against water for 2 weeks with water changes once per day. At the end of this process the particles appeared less soluble in water so they were further dialysed (tubing Mw cut off 8k) against aqueous NaOH (pH 12) for two days before dialysis against pure water for 1 day. The particles were then freeze dried to yield a purple powder.

Synthesis of iron oxide nanoparticles, DOS-FeO NPs:

The ligand was synthesized as follows: 6-Bromohexanoic acid (1 g, 5.13 mmol) was dissolved in dichloromethane (20 ml.) and N-hydroxysuccinimide (0.59 g, 5.13 mmol) added. The mixture was then cooled to 0°C followed by the addition of Ν,Ν'-dicyclohexylcarbodiimide (1.27 g, 6.16 mmol) in dichloromethane (10 niL). This was stirred at 0°C for 15 min followed by the addition of 4-(dimethyiamino)pyridine (0.125 g, 1.02 mmol). The mixture was stirred at 0°C for 1 h and then allowed to warm to room temperature where it was allowed to stir for a further 24 h. The formed precipitate was removed and the supernatant dried. The crude mixture was dissolved in dimethylformamide (DMF) (5 mL) and the precipitate removed, A yellow waxy substance was obtained after drying (1.388 g) as the crude activated acid. Dopamine hydrochloride (1.15 g, 6.06 mmol) was dissolved in dry DMF (10 mL) and degassed with nitrogen for 10 min. Diisopropylethylamine (DIPEA) ( 1 .30 g, 0.01 mol) was then added slowly and the mixture stirred for 5 min. The crude activated acid (1.388 g, 5.04 mmol) was then added and the solution stirred at room temperature for 64 h. The DMF was then removed and the residue dissolved in ethyl acetate and washed against 1 M HC1 and then brine. The organic fraction is then dried using Na₂SO₄ and concentrated to dryness. Column chromatography using ethyl acetate/hexane (3: 1) afforded the pure product. The purified material (0.32 g, 0.969 mmol) was mixed with sodium sulphite (0.367 g, 2.91 mmol), and a catalytic amount of benzyl triethyl ammonium bromide in mcthanol/water (4:9). The mixture was then refluxed overnight. The methanol was then removed and the water washed against diethyl ether. The water fraction was collected and concentrated to dryness. The pure product was then extracted from the solid using hot methanol. The nanoparticles were synthesized as follows: FeCb (1.081 g, 4 mmol), FeC12 (397.62 mg, 2 mmol), oleic acid (4.519 g, 16 mmol) were mixed in ethanol (12 mL)/degassed H?0 (9 mL)/toluene (21 mL) and the mixture refluxed at 74°C. After 1 h sodium hydroxide (620 mg, 15.5 mmol) dissolved in ethanol (1 .33 mL)/degassed H2O (1 mL)/toluene (2.33 mL) was added quickly. Reflux was continued for a further 2 h. Once cooled an excess of ethanol was added and the precipitate collected by centrifugation at 4000 g. The precipitate was washed 4 times with ethanol and the purified nanoparticles were stored at -20°C until needed.

The ligand exchange procedure was performed as follows: oleic acid functionalised iron oxide nanoparticles were dissolved in chloroform at 10 mg/mL by sonication. Separately the sulfonated catechol (CHS) ligand (20 mg) was dispersed in methanol with sonication and heating. An aliquot of 500 μL, of this mixture was added to the nanoparticles solution until complete precipitation occurred. This precipitate was collected using a magnet and the supernant removed. The precipitate was then dispersed in a methanol solution containing 30 mg of sulfonated catechol (CHS) with sonication. Sonication was continued until all the precipitate had been dissolved and the mixture was incubated for 48 h at room temperature. The now sulfonated iron oxide nanoparticles were then precipitated with an acetone/hexane mixture and the precipitate collected by magnet. This was then washed a further 3 times by dissolving in a small amount of water followed by precipitation with acetone/hexane. The precipitate was then dried to give the final sulfonated iron oxide nanoparticles. Cell Culture

HeLa (human cervical carcinoma cell line), HEK 293T (human embryonic kidney), CHO-K1 (Chinese hamster ovary cell line), Vero (African green monkey fibroblastoid kidney cells) and HT-1080 (human fibrosarcoma cell line) were purchased from ATCC (American Type Culture Collection, Rockville, MD). HEK 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Euroclone) containing 10% South American Foetal Bovine Serum (FBS-SA), 1% L-glutamine and 1% penicillin/streptomycin. HeLa cells were cultured in Eagle's minimal essential medium (MEM - Invitrogen, Carlsbad, CA) supplemented with 10% FBS-SA, 1% L- glutamine and 1% penicillin/streptomycin (Euroclone). CHO-K 1 were cultured in Ham's F12-K. medium (Invitrogen) supplemented with 10% FBS-SA, 1% L-glutamine and 1% penicillin/streptomycin. Vero cells, were grown in MEM medium (Gibco/BRL, Gaithersburg, MD) supplemented with 10 % heat inactivated foetal calf serum (FCS; Gibco-BRL), and 1 % antibiotic-antimycotic solution (Zell Shield, Minerva Biolabs GmbH, Berlin, Germany). 293TT cell line, derived from human embryonic kidney cells transformed with the simian virus 40 (SV40) large T antigen, was cultured in DMEM (Gibco-BRL, Gaithersburg, MD) supplemented with heat inactivated 10 % FCS (Gibco- BRL), Glutamax-I 1 % (Invitrogen, Carlsbad, CA) and nonessential amino acids 1% (Sigma Aldrich, Steinheim, Germany). All cells lines were grown in humidified atmosphere with 5% of C0₂ at 37°C.

Viruses

Production and purification of Lentivirus (LV-VSV-G)

Lentiviras (LV), derived from the human immunodeficiency virus (HIV), is the most widely used viral vector for gene delivery due to its ability to mediate potent transduction and stable expression into dividing and non-dividing cells. Lentiviral particles can be engineered to bear different glycoproteins (GPs) derived from other enveloped viruses (pseudotyping) allowing to an extension of the tropism. Lentiviral vectors pseudotyped with the envelope glycoprotein of Vesicular Stomatitis Virus (VSV) were used herein. Production of such phenotypically mixed virus particles was accomplished in three steps : 1) calcium phosphate transient co-transfection of HEK 293 T cells with all necessary plasmids, 2) concentration of viral vectors using PEG 6000 and 3) purification by ultracentimgation on a sucrose cushion (Tiscornia et al. 2006). Generation of infectious lentiviral particles required the expression of essential genes in HEK 293T cells through several plasmids : 1) lentiviral expression plasmid (pRRLSIN.ePTT.PGK-GFP.WPRE), carrying the transgene sequence encoding GFP, 2) plasmids encoding packaging proteins (pMDLg/pRRE, pRSV-Rev), with gag, pol, rev and tat genes and finally 3) pseudotyping plasmid (pMD2.G) encoding the heterologous envelope glycoprotein from VSV. Newly formed lentiviral particles were collected 48 hours after transfection, concentrated by precipitation using PEG-it™ (System Biosciences, SBI), resuspended in PBS and transferred in cryo-tubes and stored at -80°C. Subsequently, the titer of the virus was calculated as the number of functional particles able to deliver their genetic materials in cells (transducing units/ml - TU/ml), determined in HeLa cells through serial dilutions of lentiviral preparation, evaluating the percentage of GFP positive cells by flow cytometry (Tiscornia et al. 2006). In order to obtain lentiviral stocks with higher degree of purity, lentiviral particles stored at -80°C were rapidly thawed and further purified through ultracentrifugation on 20% (w/v) sucrose cushion. Viral preparations (200 μl) were layered on 1.5 ml of 20% sucrose in PBS into ultracentrifuge tubes (Beckman Coulter Inc). Subsequently, samples were ultracentrifuged at 19000 rpm for 2h at 20°C using a Beckman SW41 (Beckman Coulter Inc) swinging bucket rotor. Finally, the supernatant was discarded and the virus pellet was resuspended in 50 μΐ of PBS. Adenovirus (Ad)

Purified human recombinant Adenovirus (Ad) type 5 encoding for green fluorescent protein (GFP) was purchased from vector Biolabs (Philadelphia, PA, USA). According to manufacturer's data, Adenovirus was centrifuged using two sequential caesium chloride (CsCl) gradients, resuspended in PBS with 5% (w/v) glycerol, tested for sterility and titrated with UV spectrophotometric measurement at 260 nm calculating the number of viral particles (vp/ml) that resulted to be 5xl0⁸ vp/ml. Adenovirus was stored at at -80 °C.

Adeno-Associated Virus (AAV)

Different serotypes of recombinant Adeno-associated viruses (AAVs) encoding GFP (AAV2- GFP, AAVS -GFP) were purchased from Vector Biolabs (Philadelphia, PA, USA). They were stored at -80 °C in PBS with 5% glycerol.

Herpes Simplex Virus type 1 and type 2 (HSV-1 and HSV-2)

Clinical isolates of HSV-1 and HSV-2 were kindly provided by Prof. M. Pistello, (University of Pisa, Italy). HS V- 1 and HSV-2 strains were propagated and titrated by plaque assay on Vero cells.

Respiratory Syncytial Virus (RSV)

RSV strain A 2 (ATCC VR- 1540) was propagated in HEp-2 cells by infecting a freshly prepared confluent monolayer grown in MEM supplemented with 2% of FCS. When the cytopathic effect involved the whole monolayer, the infected cell suspension was collected and the viral supernatant was clarified. The virus stocks were aliquoted and stored at -80°C. The infectivity of virus stocks was determined on HEp-2 cell monolayers by standard plaque assay. The cell lines and the RSV were obtained from the American Type Culture Collection (Manassas, VA, USA).

Dengue virus type 2 (DENV-2)

Dengue virus type 2 was obtained from Dr. Jochen Bodem, University of Wurzburg (Wurzburg,

Germany), propagated and titrated by immuno-stained plaque assay in Vero cells. Human Papilloma Pseudovirus (HPV16 PsV) production

Plasmids and 293TT cells used for pseudovirus (PsV) production were kindly provided by John

Schiller (National Cancer Institute, Bethesda, MD). HPV-16 PsVs were produced according to previously described methods (Buck et al, 2005). Briefly, 293TT cells were transfected with plasmid expressing the papillomavirus major and minor capsid proteins (L1 and L2, respectively), together with a reporter plasmid expressing the secreted alkaline phosphatase (SEAP), named pYSEAP. Capsids were allowed to mature overnight in cell lysate; the clarified supernatant was then loaded on top of a density gradient of 27 to 33 to 39 % Optiprep at room temperature for 3 h. The material was centrifuged at 28000 rpm for 16 h at 4°C in an SW41.1 rotor (Beckman Coulter, Inc., Fullerton, CA) and then collected by bottom puncture of the tubes. Fractions were inspected for purity in 10% sodium dodecyl sulfate (SDS)-Tris-glycine gels, titrated on 293TT cells to test for infectivity by SEAP detection, and then pooled and frozen at - 80 °C until needed. The L1 protein content of PsV stocks was determined by comparison with bovine serum albumin standards in Coomassie-stained SDS-polyacrylamide gels. Cytotoxicity Assay

The toxicity of Au-NPs was examined using propidium iodide (PI) flow cytometric assay or MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3 carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H- tetrazolium] assay. Propidium iodide, a fluorescent stain for nucleic acid, allows to determine the amount of dead cells due to its inability to penetrate into live cells. Indeed, PI could only penetrate into cells that exhibit compromised plasma membrane and intercalates into double stranded DNA. Once the dye is bound to DNA it is excited at 488nm and emits at a maximum wavelength of 617 nm. The percentage of viable cells was measured following the same experimental conditions as for the NPs/virus interaction study.

Cells were incubated with different concentrations of NPs (from 0.1 μg/ml to 100 μg /ml) for 48 h at 37°C. After incubation with nanomaterials, cells were harvested by trypsin, washed with 1ml of PBS supplemented with 1% BSA and the pellet was resuspended in 500 μΐ of PBS. Finally, 2 μΐ of PI, at the concentration of 50 μg/ml in PBS, were added to the samples and incubated for 5 min at RT in the dark. PI fluorescence was immediately determined on a FACS CANTOII flow cytometer (Beckton Dickinson, San Jose, CA). Unstained cells were used as negative control samples. The results were analysed using BD FACSDiva software. For the MTS assay, cell cultures seeded in 96-well plates were incubated with different concentrations of Au-NPs or ligand under the same experimental conditions as described for the antiviral assays. Cell viability was determined by the CellTiter 96 Proliferation Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Absorbance was measured using a Microplate Reader (Model 680, BIORAD) at 490 run. The effect on cell viability at different concentrations of nanoparticles was expressed as percentage of live cells, by comparing the absorbance of treated cells with the one of cells incubated with culture medium.

Au-NPs preparation for antiviral assay

NPs were dissolved in Milli-Q grade water, sonicated for 20 min at room temperature and then filtered with a 0.22 μηι filter in order to sterilize and remove precipitates. Before the use with cells, an appropriate amount of 1 Ox PBS was added to the NPs solutions in order to obtain the final concentration ready to use in 1x PBS. Inhibition Assays LV-VSV-G, Adenovirus and AW2 and AVV5

VSV-G pseudotyped lentivirus (LV-VSV-G) or Adenovirus (ADS) or Adeno Associated Viruses (AAV2 AAV5), carrying GFP as reporter gene, were resuspended in PBS and incubated with increasing concentrations of sulfonated or non-sulfonated NPs in PBS for 1h at 37°C prior to cell infection. The mixture of virus/nanoparticles was subsequently added to HeLa cells for LV- VSV-G and AD5 whereas CHO-Kl cell lines were used for both serotypes of A AVs (AAV2 and AAV5). Transduction efficiency, calculated as the % GFP+ cells, of LV-VSV-G and Adenovirus was measured flow cytometry while confocal laser scanning microscopy was used to for AAVs. Transduction was stopped after 48h and cells were fixed with 1% p-formaldehyde (PFA) for 10- 15 minutes at room temperature and resuspended in PBS.

Inhibition Assay HSV-1, HSV-2

The effect of Au-NPs on HSV infection was evaluated by a plaque reduction assay. Vero cells were pre-plated 24 h in advance in 24- well plates at a density of 10⁵ cells. Increasing concentrations of nanoparticles or ligand were incubated with HSV-1 or HSV-2 (multiplicity of infection (MOI) 0.0003 plaque forming units (pfu)/cell) at 37 °C for 1 hour and then the mixtures were added to the cells. Following virus adsorption (2 h at 37°C), the virus inoculum was removed, the cells were washed with medium and then overlaid with a medium containing 1.2 % methylcellulose. After 24 h (HSV-2) or 48 h (HSV-1 ) of incubation at 37°C, cells were fixed and stained with 0.1 % of crystal violet in 20 % ethanol and viral plaques were counted. The concentration producing 50 % reduction in plaque formation (IC₅₀) was determined using the Prism software by comparing drug-treated and untreated wells.

Inhibition Assay HPV-16 PsV

293TT cells were preplated 24 h in advance in 96-well tissue culture-treated flat bottom plates at a density of 2xl0⁴ cells/well in 100 of neutralization buffer (DMEM without phenol red, 10 % FBS, 1 % glutamate, 1 % nonessential amino acids, 1 % penicillin-streptomycin-fungizone, and 10 mM HEPES). Diluted PsV stocks (80 μL/well) were placed on 96-well non treated sterile, polystyrene plates (Nalge-Nunc, Roskilde, Denmark), combined with 20 μL, of serially diluted nanoparticles or ligand, and placed for 1 h at 37°C. The 100- μL PsV-compound mixture was transferred onto the pre-plated cells and incubated for 72 h. The final concentration of PsV was approximately 1 ng/mL L1. After incubation, 25 μL, of supernatant was harvested. The SEAP content in the supernatant was determined using a Great Escape SEAP Chemilumincscence Kit (BD Clon-tech, Mountain View, CA) as directed by the manufacturer. 30 min after the addition of the substrate, samples were read using a Wallac 1420 Victor luminometer (PerkinElmer Life and Analytical Sciences, Inc., Wellesley, MA).

Inhibition Assays RSV

Nanoparticles or ligand were serially diluted and incubated with RSV (MOI 0.01) for 1 h at

37 °C. Then the mixture was added to lxlO⁴ A549 cells grown as monolayers in a 96-well plate to allow the viral adsorption for 3 h at room temperature; the monolayers were then washed and overlaid with 1.2 % methylcellulose medium. Three days post-infection, cells were fixed with cold methanol and acetone for 1 min and subjected to RSV-specific immunostaining using an RSV monoclonal antibody (Ab35958; Abeam, Cambridge, United Kingdom). Immunostained plaques were counted, and the percent inhibition of virus infectivity was determined by comparing the number of plaques in treated wells with the number in untreated control wells.

Inhibition assay DENV-2

Two fold dilution of NPs were added to DENV-2 (MOI 0.03) and incubated for 1 h at 37 °C in

5% C0₂. After incubation the NPs/virus mixture was added to 20,000 Vera cells pre-plated day before in DMEM with 2% fetal bovine serum, 100 U of penicillin/ml and 100 μg of streptomycin/ml. After 72 h incubation at 37 °C in 5% C02, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. The cells were washed with IX PBS and incubated overnight at 4 °C with dengue virus type 2 sero type-specific mouse monoclonal antibody, which was harvested from HB-46 cells (ATCC, Manassas, VA). Then, the wells were washed three times with IX PBS and incubated 90 min with Cy3-labeled donkey anti mouse IgG (Jackson Immunoresearch Europe, Suffolk, UK). Infected cells were documented using fluorescence microscope Olympus IX-81 (Hamburg, Germany). ImageJ software (NIH) was used for image analysis and evaluation of percentage of infected cells. NPs concentrations required to inhibit virus replication by 50% (EC50) were calculated using nonlinear regression analysis using Graph Pad Prism, version 6.05 (GraphPad Software, La Jo!la, CA).

Evaluation of virucidal activity against LV-VSV-G

The effect of NPs upon transduction on HeLa cells of recombinant LV-VSV-G (10⁶ TU/ml - MOI 10) and Adenovirus (2x10⁷ TU/ml - MOI 20) was evaluated by incubating an effective inhibitory concentration of MUS:OT-NPs (1000 μg/ml) with viruses for 2h at 37°C as previously described (Shogan, 2006). After incubation, transduction efficiency was determined by titration at high dilutions and calculated as the percentage of GFP+ cells by flow cytometry.

Evaluation of virucidal activity against HSV-2, RSV, HPV-16

Viruses (10⁵ pfu for HSV-2 and HPV-16, 10⁴ for RSV) and 100 μg/ml of MUS:OT-NPs were incubated at different time points (0, 5, 30, 60 or 120 min) at 37°C and then the virucidal effect was investigated with serial dilutions of the mixtures. Viral titers were calculated at dilutions at which the nanoparticle was not effective. Evaluation of virucidal activity against DENV-2

Dengue virus type 2 was mixed with 200 μg/ml of all MUS PUl and incubated for 2 h. As a control same amount of virus was incubated without NPs. After 2 h incubation residual virus was diluted twofold, each dilution was added to 20,000 Vero cells in triplicate, incubated for 3 days in 5% C0₂, 37°C and titer was determined by immuno-stained plaque assay.

Viral Yield Reduction Assay

The assay is finalized to quantify the antiviral effect of compound testing its effect on the production of infectious viruses. Vero cells were seeded in 24-well plates at a density of 10⁵ cells/well and infected in duplicate with HSV-2 at a multiplicity of infection (MOI) of 0.01 plaque-forming units (pfu/cell) and in the presence of serial dilutions of the compound. Following adsorption at 37°C for 2 h, the virus inoculum was removed and cultures were grown in the presence of serial dilutions of MUS:OT-NPs until control cultures displayed extensive cytopathology. Supernatants were harvested and pooled as appropriate 24-48 h after infection and cell-free virus infectivity titers were determined in duplicate by plaque assay in Vera cell monolayers. The end-point of the assay was the effective concentration of nanoparticle that reduced virus yield by 50% (EC₅₀) compared to untreated virus controls.

FACS analysis

Cells were trypsinized, washed with PBS and fixed in 1% PFA (paraformaldehyde) in PBS for 10 min at room temperature. Approximately 2xl0⁴ events (cells) were analysed per sample and cells with no virus were used as negative control in order to determine the background of autoiluorescence. Transduced cells were calculated as the percentage of GFP+ cells over the total population of analysed cells. The expression of the GFP protein on cells transduced with LV-VSV-G or AD5 was assessed through the BD FACSCalibur™ flow cytometer (BD Biosciences) and data were analysed with BD CELLQuest™ software (BD Biosciences).

Confocal Laser Scanning Microscopy (CLSM)

Transduction efficiency, as the number of GFP+ cells, of AAVs (AAV2, AAV5), in presence or absence of MUS:OT-NPs, was visualized by using an inverted confocal laser scanning microscope (Leica TCS SP5) equipped with blue (488 nm) excitation laser line, objectives 40x and 63x. Cells were previously fixed in 2% of p-fonnaldehyde (PFA) in PBS for 10 min at room temperature.

TEM HSV-2 and HPV-PsV (10⁵ pfu) were incubated with only medium, or with 100 μg/mL Au-NPs (MUS:OT, EG2-OH) was allowed to adsorb for about 5 min on carbon- and Formvar-coated grids, and then the grids were rinsed several times with water. Grids were negatively stained with

0.5% uranyl acetate, and excess fluid was removed with filter paper. Observations and photographs were made using a CM 10 electron microscope (Philips, Eindhoven, The Netherlands). Images were adjusted for brightness and contrast with GIMP software (GNU Image Manipulation Program).

Cryo-TEM

For cryo-TEM HSV-2 and HPV-PsV (10⁵ pfu) treated with Au-NPs (MUS:OT, EG2-OH) 100 μg/mL suspended in buffer were flash-frozen in their native hydrated state. Briefly, a droplet has been deposited onto a holey carbon grid (Quantifoil® Micro Tools Gmbh, Jena, Germany), blotted and subsequently vitrified in liquid ethane using an FEI vitrobot Marc IV. Imaging was performed at - 175 C in a FEI Tecnai Spirit BioTWIN 80kV transmission electron microscope at magnifications of 30.000-48. OOOx in LowDose Mode (approx. 1-3 electrons/Angstrom²).

Agarose Gel Electrophoresis

VSV-G pseudotyped lenti virus was purified through ultracentrifugation on 20% (w/v) sucrose cushion. Viral preparation (200 μL,) of unpurificd LV- VSV-G (~108 particle/mL) in PBS were layered on top of 1.5 niL of 20% sucrose solution in PBS into ultracentrifuge tubes (Beckman Coulter Inc). Ultracentrifugation was performed with swinging bucket rotor SW41 (Beckman Coulter Inc.) at 19.000 rpm for 2 h at 25°C. Then supernatant was removed, and 50 of PBS were added to the virus pellet. Before resuspension, the viral pellet was equilibrated for 15 min on ice bath. Generally, 10 μL, of purified LV- VSV-G diluted 1 to 2 were loaded onto a 0.8 wt % agarose gel dissolved in TBE buffer, added with Gel Red™ (Biotium) DNA staining. Run was performed at 100 V for 30-45 minutes. After running, an image of the gel was acquired under UV lamp in order to evaluate the presence of the NPs as a defined band or a smear (brownish-red colour). Finally, protein staining was performed with InstantBlue™ solution (Expedeon) over night, under rocking. The gel was washed several times in MilliQ water, until the aspecific blue staining was removed.

Data analysis

All results are presented as the mean values from three independent experiments. The EC50 values for inhibition curves were calculated by regression analysis using the program GraphPad Prism version 5.0 (GraphPad Software, San Diego, California, U.S.A.) to fit a variable slope- sigmoidal dose-response curve. The selectivity indexes SI were calculated dividing the CC50 for the EC50.

Calculation of molecular weight of gold nanoparticles

The calculations of the molecular weight of the NPs followed the equations described in literature NPs are composed of a gold core and a monolayer of organic molecules arranged onto NPs surface. A reasonable estimation of the molecular weight of NP is made by following equations:

where NA is the Avogadro's number, the density of gold bulk is 19.32 ² and the

density of the ligand is 1.2 The length of MUS is approximately 1.7 nm, the

radius of MUS:OT NP, r_c is 1.4 nm. The molecular weight, M!F of MUS:OT NP is 2.2x10⁵ Da.

The number of NP in 1 mg is 3xl0¹⁵. Molecular dynamics simulations of a nanoparticlc-virus complexation

Atomistic molecular dynamics (MD) simulations of ligated gold nanoparticles (NPs) adsorbed on viral capsids was performed. The modelled NPs have cores of different diameters (2.4 nm and 5 nm). Two types of ligands (OT, MUS) in a 1 : 1 ratio were evenly distributed on the surfaces of NPs (density ~ 5 ligands per nm2). The adsorption of these NPs on capsid segments of HPV and dengue viruses was modelled. The structures of viral capsid segments were based on pdb IDs 3J6R (HPV-16)⁴ and 1P58 (dengue) . The systems were described within CHARMM general and protein force fields. The simulations were performed with NAMD software in an NPT ensemble, using the Langevin dynamics and a timestep of 2 fs. Nonbonding interactions were calculated using a cut-off distance of 10 A and long range electrostatic interactions were calculated by the PME method in the presence of periodic boundary conditions. In the simulations, NPs were placed close to the capsid surface and solvated in a 0.15 M NaCl solution.

Molecular Dynamics Simulations

Coupling of functionalized NPs with HPV and Dengue viruses were simulated.

HPV virus:

In the capsid of an HPV virus, the K278, K356, K361, K54 and K59 lysine (positively charged) residues of L I proteins are mainly responsible for a specific binding to (negatively charged) HSPG^{1 1}'¹². Fig. 3 presents two examples of NPs interacting with L1 proteins forming the HPV capsid, which were examined in a physiological solution. The dynamics of these systems is captured by movies data, showing 1) how a small NP (2.4 nm core) binds to a single L1 protein pentamer via HSPG-specific binding sites and 2) how a large NP (5 nm core) binds to two L1 pentamers and induces changes in their orientations. Besides the specific binding of NPs to the HSPG binding sites, in a later stage of NP-HPV binding, the charged NPs could also bind to capsid proteins in other possible ways. Figures 18A and 18B show that the bottom of L1 protein is a highly positively charged, which may be attractive for (negatively charged) NPs binding. Fig. 18C shows this binding which could occur when the L1 protein is reoriented by the NPs. The simulations show that this binding can be very strong.

Dengue virus:

Coupling between the above described NPs and a dengue virus capsid was modeled (Fig. 19A). The simulated system contained five major envelope protein E units from the viral capsid, which form the shape of a star (Fig. 19B). The modelled segment of the dengue capsid contained a high local concentration of HSPG binding amino acid residues (K305, K307, K310, K295, K291 , R288, R286, K284, R188, K388, K393 and K394) , which are primarily positively charged lysine and arginine residues. Initially, the NP was placed close to the HSPG binding sites and simulated the systems for 10 ns. The simulations, presented in Fig 19C, show that the number of interacting points between the amino acids and sulfonate groups gradually increases. Eventually, 5-6 amino acids interact with the NPs sulfonate groups, which generates large deformation forces on the capsid. Figure 20 provides additional binding sites between the NPs and the capsid proteins, eventually used at later times. A system containing six major envelope protein E units from the viral capsid, forming the shape of the leaf, is shown in Fig. 20B,C. Examining the electrostatic properties of this leaf-shaped capsid segment reveals that capsid proteins have a dipolar nature. A strong positive potential is present in the central convex part of the capsid segment (vims exterior) regions, while a negative potential is present at the concave side of the capsid segment (virus interior). The positively charged central part of the envelope protein can be attractive for an additional binding to (negatively charged) NPs and possible wrapping around its surface. Together with the specific binding of the NPs to the capsid, these additional (less specific) binding events could ultimately lead to the capsid disintegration.

Capsid proteins could also chemically change in response to the changes in their environment, such as pH or accumulated charges. For example, capsid proteins of many viruses, including dengue virus, can become more positively charged through histidine protonation, which is an important part of the pH sensing mechanism of many viruses. Protonation of pH sensing histidines can lead to a response in the form of large conformational changes in capsid proteins. In the case of a dengue vims capsid, two of the pH sensing histidine residues are close to the heparan sulfate recognition amino acids, all of which lie at the interface of capsid proteins. In response to the presence of supercharged NP, the pH sensing histidines could become protonated, which can destabilize the capsid structure by making it more prone to capsid protein reorientation. However, as most of the other key pH-sensing histidine residues are further away from the NP-binding region of the capsid protein, this mechanism is likely to play a minor role in the capsid disintegration.

Claims

Claims

1. A virucidal metallic nanoparticle comprising multiple alkyl sulfonate ligands that provide the attachement receptor for viruses, and optionally multiple additional alkyl ligands, wherein

the alkyl sulfonate ligand is

, and wherein

Z is absent or selected from the group comprising O, S, and

y is 5 to 20,

X is substituted or unsubstituted cyclo-C₅-C₇-alkyl or aryl, wherein one or more substituents are selected from the group comprising C₁-C₄-alkyl and -OH. and the optional additional alkyl ligand is -Q-CH₂-(CH₂)w, and wherein

Q is absent or selected from O or S,

w is 4 to 20.

2. The virucidal metallic nanoparticle of claim 1 , wherein

wherein X is aryl substituted with two -OH,

y is 5 to 1 1.

3. The virucidal metallic nanoparticle of any one of claims 1-2, wherein the alkyl sulfonate ligand is 1 1 -mercapto- 1 -undecanesulfonate (MUS) or 6-((3,4- dihydroxyphenethyl)amino)-6-oxohexane-l -sulfonate (DOS).

4. The virucidal metallic nanoparticle of any one of claims 1 -3, wherein the additional alkyl ligand is octalen thiol (OT).

5. The virucidal metallic nanoparticle of any one of claims 1 -4, wherein the metallic nanoparticle is gold nanoparticle or iron oxide nanoparticle.

6. The virucidal metallic nanoparticle of any one of claims 1-5, wherein the ratio between alkyl sulfonate ligands and alkyl ligands is from 1.5: 1 to 2.5: 1.

7. The virucidal metallic nanoparticle of any one of claims 1-6, wherein the ratio between alkyl sulfonate ligands and alkyl ligands is about 2: 1.

8. The virucidal metallic nanoparticle of any one of claims 1 -7, wherein the alkyl sulfonate ligands are the same or different.

9. The virucidal metallic nanoparticle of any one of claims 1 -8, wherein the alkyl ligands are the same or different.

10. A pharmaceutical composition comprising an effective amount of one or more virucidal metallic nanoparticles of any one of claims 1 -9 and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

1 1. The virucidal metallic nanoparticle of any one of claims 1-9 for use in treating and/or preventing viral infections and/or diseases associated with viruses.

12. The virucidal metallic nanoparticle for use in treating and/or preventing viral infections and/or diseases associated with viruses of claim 1 1 , wherein the viruses are HSPG binding viruses.

13. The virucidal metallic nanoparticle for use in treating and/or preventing viral infections and/or diseases associated with viruses of claim 12, wherein the viruses are selected from the group comprising herpes simplex virus (HSV), human papillomavirus virus (HPV), respiratory syncytial virus (RSV), dengue virus and lenti virus (a human immunodeficiency virus (HIV) derived virus).

14. A virucidal composition comprising an effective amount of the virucidal metallic nanoparticles of any one of claims 1 -9 and optionally at least one suitable carrier.

15. A method of disinfection and/or sterilization comprising using the virucidal composition of claim 14.

16. A device comprising the virucidal composition of claim 14 or the virucidal metallic nanoparticles of any one of claims 1-9 and means for applying or dispensing the virucidal composition or the virucidal metallic nanoparticles.

17. A use of the virucidal metallic nanoparticles of any one of claims 1 to 9 or the virucidal composition of the claim 14 for sterilization and/or for disinfection.