WO2014147287A1

WO2014147287A1 - Nanocrystalline cellulose (ncc) as an antiviral compound

Info

Publication number: WO2014147287A1
Application number: PCT/FI2013/050979
Authority: WO
Inventors: Jukka Seppälä; Justin ZOPPE; Kristiina JÄRVINEN; Ari Hinkkanen; Ville RUOTTINEN
Original assignee: Jukka Seppälä; Zoppe Justin; Järvinen Kristiina; Ari Hinkkanen; Ruottinen Ville
Priority date: 2013-03-21
Filing date: 2013-10-10
Publication date: 2014-09-25

Abstract

The present invention concerns nanocrystalline cellulose (NCC) as an antiviral compound, methods for the preparation of such NCC, antiviral compositions of such NCC together with a carrier, and uses of such NCC for medical or disinfections purposes.

Description

NANOCRYSTALLINE CELLULOSE (NCC) AS AN ANTIVIRAL

COMPOUND

Technical Field

The present invention relates to nanocrystalline cellulose (NCC) as an anti-viral compound. The invention also relates to a process for producing NCC, and the use of NCC for reducing the risk of viral infections, or for treating or preventing viral infections. In particular, the invention relates to a composition of such NCC, for reducing the risk of transmission, or treating or preventing viral infections; to a process for producing such a composition; and the use of such a composition.

Background Art

Viral infections are causing debilitating diseases in humans in wide areas of the world and therefore discovery of effective, cheap and stabile formulations used as prophylactic or incidence medicine in endemic areas is valuable. Numerous therapeutic and preventative compounds are available and in development, for inhibition of viruses, especially human immunodeficiency virus (HIV) [Teixeira, C. et al., Eur. J. Med. Chem. 2011, 46, 979-992]. One family of inhibitors are based on polyanionic compounds which prevent virus binding to target cells via electrostatic interactions with the viral envelope [Pirrone, V. et al., Antiviral Res. 2011, 90, 168- 182]. These include water-soluble anionic polymers such as polystyrene sulfonate and polysaccharides, such as cellulose sulfate and cellulose acetate phthalate, which have a long history of antiviral activity. These polyanionic compounds are effective against many viruses in vitro, such as human immunodeficiency virus (HIV), herpes simplex virus (HSV), influenza and human papillomavirus (HPV). Polysulfated compounds were cautioned against systemic administration due to interference with blood coagulation [Flexner, C. et al., Antimicrob Agents

Chemother 1991, 35, 2544-50], however they were suggested to be suitable active ingredients of topical microbiocides. Since cellulose sulfate (Ushercell) was patented as an antimicrobial and contraceptive agent [Anderson, R. A. et al., WO 9712621, 1997 and Usher, T. C. et al., WO 2002002189, 2002], it proceeded to phase III clinical trials for the prevention of vaginal HIV transmission, however trials were terminated due to lack of effectiveness [Van Damme, L. et al., N. Engl. J. Med. 2008, 359, 463-472]. This could later be explained by observations that at low concentrations cellulose sulfate stimulated viral replication, whereas at higher concentrations it was inhibitory, which poses a challenge for dosing in different clinical settings. Due to the high molecular weight (>500,000 g/mol) and high degree of sulfation (12-18 %) of cellulose sulfate, it cannot be administered parenterally. Accordingly, there is a need for alternative polysulfated compounds with low molecular weight (<50,000 g/mol), low degree of sulfation (2-7 %) and low non-specific cellular uptake which can be administered topically and parenterally, e.g. via subcutaneous injection.

Anionic acid groups in proteins, such as sulfated tyrosines, have a critical role in binding events of biological systems; for example, they facilitate the HIV-1 virus entry into host T-cells and macrophages [Stone, M. J. et al., New Biotechnol. 2009, 25, 299]. Systems that successfully mimic these interactions for HIV inhibition include nanoparticles with sulfate groups [Di Gianvincenzo, P. et al., Bioorg. Med. Chem. Lett. 2010, 20, 2718] and drug-like small molecules containing phenyl sulfonate groups [Acharya, P. et al., ACS Chem. Biol. 2011, 6, 1069-1077], which are termed tyrosine sulfate mimetics. Powdered forms of cellulose have a long history of use in the pharmaceutical industry. Powdered cellulose is used as an excipient in various dosage forms, including tablets, two-piece hard capsules, soft capsules, aqueous suspensions for per oral delivery and suppositories [Rowe, R. C. et al. (editors), Handbook of Pharmaceutical Excipients, Pharmaceutical Press and American Pharmacists Association, 2012]. Typically, the form of cellulose that is termed microcrystalline cellulose (MCC) is used in tablets. MCC is purified, depolymerized alpha-cellulose derived from plant sources [O'Connor, R. E. et al., Pharm. Res. 1993, 10, 356-61]. MCC is generally recognized as safe (GRAS) by the US Food & Drug

Administration [FDA SCOGS database; Report No. 25, 1973; Accessed February 5, 2013]. On the other hand, nanocrystalline cellulose (NCC) is extracted from cellulosic biomass by acid hydrolytic extraction processes. Nanocrystalline cellulose (NCC), also known as cellulose nanocrystals (CNCs) or cellulose whiskers, have a very high surface area to volume ratio due to the nanometer size of the rod-shaped crystals. NCC is non-cytotoxic [Lam, E. et al., Trends Biotechnol. 2012, 30, 283-290, Jackson, J. K. et al., Int. J. Nanomed. 2011, 6, 321-330 and Dong, S. et al., Nano LIFE 2012, 2, 1241006], and has a well-established biocompatibility due to its inert chemical nature. NCC produced from acid hydrolysis can form highly stable aqueous dispersions as a result of anionic functional groups on the crystal surfaces. Thus, NCC offers a naturally abundant nanoparticle that can act as an anti- viral compound. The use of NCC as a substrate for controlled release of drugs such as tetracycline, doxorubicin, docetaxel and paclitaxel has been recently patented [Burt, H. M. et al., WO 2012068670, 2012]. However, there are no reports covering the use of NCC as an anti-viral compound.

US 8309708 B2 concerns crystalline sulfated cellulose II and its production.

Crystalline sulfated cellulose II referred to in this patent is derived from the acid soluble polysaccharides (residual stream) after sulfuric acid hydrolysis of native cellulose I. The said sulfated cellulose II is regenerated from solubilized fractions and has a molecular weight < 10,000 g/mol. In the present invention to be described below, the acid insoluble rod-shaped nanocrystalline cellulose (NCC) is in the native cellulose I form with molecular weight of >15,000 g/mol. In addition, only the surface of NCC, rod-shaped nanoparticles, is derivatized with sulfate groups.

US 2007014812 Al describes cellulose-based anionic polymers which are cellulose derivatives with 32-35 wt.% substitution. In addition, the starting materials utilized for synthesis of cellulose-based anionic polymers are commercially available cellulose derivatives such as cellulose acetate phthalate (CAP), cellulose acetate trimellitate (CAT) and hydroxypropyl methyl cellulose phthalate (HPMCP). In the patent, sulfation or sulfonation of cellulose derivatives is achieved with either sulfur trioxide or anhydride chemistries of individual cellulose polymer chains. In the present invention to be described below, nanocrystalline cellulose (NCC), rod- shaped nanoparticles, is produced purely by sulfuric acid hydrolysis from a number of native cellulosic raw materials, such as cotton fibers or filter paper, which results in an overall degree of sulfation from 2-7 wt.%. Then, NCC can be desulfated and optionally further derivatized using e.g. epoxide and isothiocyanate chemistries. In addition, the derivatization of NCC with sulfate and phenyl sulfonate groups only occurs on the surface of the rod-shaped nanoparticles. US 20090247740 A1 describes spherical sulfated cellulose particles above 50 micron in size, such that they are not on the nano scale. In addition, spherical sulfated cellulose is produced by sulfuric anhydride esterification. In the present invention, rod-shaped nanoparticles <1 micron, termed nanocrystalline cellulose (NCC) are derived purely from sulfuric acid hydrolysis.

JP 2008266265A describes sulfated cellulose, where individual cellulose polymer chains which have been sulfate esterified at least 12% wt, which can be termed cellulose sulfate. In the present invention, rod-shaped nanoparticles <1 micron of NCC produced from sulfuric acid hydrolysis are only derivatized with sulfate groups on the surface (2-7 wt. %).

EP 2040716 B1 also describes sulfated cellulose for treatment of Rhinovirus infection, where individual cellulose polymer chains which have been sulfate esterified at least 12% wt., which can be termed cellulose sulfate. In the present invention, rod- shaped nanoparticles, < 1 micron, of NCC produced from sulfuric acid hydrolysis are only derivatized with sulfate groups on the surface (2-7% wt.).

US 7235536 B2 describes sulfated cellulose for treatment of papilloma virus infection. Here individual cellulose polymer chains, which have been sulfate esterified at least 12% wt., which can be termed cellulose sulfate and has a molecular weight >500,000 g/mol. In the present invention, rod-shaped nanoparticles,< 1 micron, of NCC produced from sulfuric acid hydrolysis are only derivatized with sulfate groups on the surface (2-7% wt).

WO 2005004882 A1 describes antiviral charged polymers. The sulfated

polysaccharides describes are carboxymethyl dextran sulfate or carboxymethyl cellulose that has been sulfate esterifed, preferably at least 13% wt. This refers to individual carboxymethyl cellulose polymer chains that have been derivatized with anionic groups. In the present invention, rod-shaped nanoparticles, < 1 micron, of NCC produced from sulfuric acid hydrolysis are only derivatized with sulfate groups on the surface (2-7 wt. %).

Summary of Invention

Technical Problem

NCC that exhibits anti-viral properties typically bears hydroxyl groups and/or anionic acid groups, such as sulfate, sulfonate, phenyl sulfate, or phenyl sulfonate groups, on the crystal surfaces. In this invention it has been demonstrated that NCC is capable of inhibiting infection by fluorescent Semliki Forest virus (SFV) vector VA7-EGFP and parental SFV rA774 virus of primate cells in vitro the magnitude of inhibition being dependent on NCC concentration and surface functionality (see Figure 1 and 3). In this invention it has also been confirmed that NCC is non- cytotoxic to human corneal epithelial (HCE) cells and African green monkey kidney (Vero B) cells in vitro (see Figure 2, 4 and 5). Semliki Forest Virus (SFV), closely related to the Chikungunya Virus (CHIK), is an arthropod-borne alphavirus found in parts of Africa and is known to cause disease in domestic animals and humans [Gibbons, D. L. et al., Nature (London, U. K.) 2004, 427, 320-325, Helenius, A. et al., J. Cell Biol. 1980, 84, 404-20, Lescar, J. et al., Cell (Cambridge, MA, U. S.) 2001, 105, 137-148 and Strauss, J. H. et al., Microbiol. Rev. 1994, 58, 491-562].

This invention seeks to provide nanocrystalline cellulose (NCC) for reducing the risk of viral infections, or for treating or preventing of viral infections. Such NCC typically bears hydroxyl groups and/or anionic acid groups, such as sulfate, sulfonate, phenyl sulfate, or phenyl sulfonate groups, on the crystal surfaces.

This invention also seeks to provide a composition comprising an effective amount of nanocrystalline cellulose (NCC) for reducing risk of viral infections, or for treating or preventing of viral infections. Such a composition typically comprises of NCC bearing hydroxyl groups and/or anionic acid groups, such as sulfate, sulfonate, phenyl sulfate, or phenyl sulfonate groups, on the crystal surfaces.

Furthermore, this invention seeks to provide a method to produce nanocrystalline cellulose (NCC) for use as antiviral agent.

Yet another object of the invention is a method to produce a composition

comprising an effective amount of nanocrystalline cellulose (NCC) for reducing the risk of viral infections, or for treating or preventing viral infections.

Still further, this invention seeks to provide the use of nanocrystalline cellulose (NCC) for reducing the risk of viral infections, or for treating or preventing viral infections. The final aspect of the invention is the use of a composition comprising an effective amount of nanocrystalline cellulose (NCC) for reducing the risk of viral infections, or for treating or preventing viral infections.

Solution to Problem

Thus, in one aspect this invention concerns nanocrystalline cellulose (NCC) as an antiviral compound.

In another aspect, the invention concerns a method for the preparation of a nanocrystalline cellulose (NCC) , in particular nanocrystalline cellulose (NCC) useful as an antiviral compound. This method is characterized by the steps of (a) treating native cellulose with a strong acid, preferably strong sulfuric acid, most preferably a 65 wt-% aqueous sulfuric acid solution, to dissolve the amorphous regions of the cellulose;

(b) separating the acid-insoluble rod shaped, sulfated nanoparticles from the dissolved amorphous cellulose;

(c) recovering the acid-insoluble rod shaped, sulfated nanoparticles; and optionally

(d) desulfating said sulfated nanoparticles, and optionally further substituting hydroxyl groups on the desulfated nanoparticles with other anionic groups. Furthermore, this invention concerns an antiviral composition comprising an antiviral compound and a carrier, wherein the antiviral compound is a

nanocrystalline cellulose (NCC) according to any of the claims 1-9.

Still further, the invention concerns an antiviral fabric useful for disinfection purposes, especially for reducing the risk of viral infections in hospitals, clinics and the like, wherein said fabric is a woven or non- woven fabric immersed with an antiviral nanocrystalline cellulose (NCC) according to any of the claims 1-9 or an antiviral composition comprising antiviral nanocrystalline cellulose (NCC) and a carrier according to claim 16.

The invention concerns also a method for preventing or treating viral infections in a mammal, said method comprising administering to said mammal in the need thereof an effective amount of an antiviral nanocrystalline cellulose (NCC) according to any of the claims 1-9.

The present invention also concerns novel uses of nanocrystalline cellulose and of fabrics containing the same as claimed in claims 20 to 24.

Advantageous Effects of Invention

The present invention describes the use of nanocrystalline cellulose (NCC) as an anti-viral compound for use alone or in conjunction with other formulations and compositions. As will be disclosed in more detail below, the invention shows the great utility of NCC for medical or disinfections purposes (such as a disinfectant) for example in the form of topical, parenteral and solid or liquid oral formulations. Brief Description of the Drawings

Figure 1 illustrates the influence of NCC derived from cotton fiber bearing sulfate groups as a function of concentration on the inhibition of SFV infection in vitro. Figure 2 illustrates the effects of NCC derived from cotton fiber bearing sulfate groups (batch 1, 2 and 3) and NCC derived from Whatman filter paper bearing sulfate groups on human corneal epithelial (HCE) cell viability in vitro after 1 hour of exposure.

Figure 3 illustrates the influence of NCC derived from cotton fiber bearing sulfate groups and NCC derived from cotton fiber modified with phenyl sulfonate groups (NCC-SPTC) (0,3 % wt.) on the inhibition of SFV infection in vitro at different SFV concentrations.

Figure 4 illustrates the effects of NCC derived from cotton fiber modified with phenyl sulfonate (NCC-SPTC) and (NCC-EBEA) groups on human corneal epithelial (HCE) cell viability in vitro after 1 hour of exposure.

Figure 5 illustrates the effects of NCC derived from cotton fiber after desulfation

(NCC-DES) on human corneal epithelial (HCE) cell viability in vitro after 1 hour of exposure.

Description of Embodiments

Nanocrystalline cellulose (NCC) herein refers to crystalline cellulose in which the crystals are of a particle size on the nano scale, i.e. from 5 nm to 1000 nm. In this respect the particle size is the dimension corresponding to the diameter of a sphere encasing the nanop article.

Nanocrystalline cellulose (NCC) is extracted as a colloidal dispersion by acid hydrolysis, typically sulfuric or hydrochloric acid, of cellulosic materials, such as cotton fiber, filter paper, wood pulp, bacteria, or sea animals called tunicates. NCC consists of cellulose, a linear polymer with β(1->4) linked D-glucose units, the chains of which are arranged in crystalline and amorphous domains. NCC is characterized by high crystallinity (~85 %) which approaches the theoretical limit of cellulose chains.

NCC obtained via sulfuric hydrolytic extraction has a molecular weight in the range of 15,000 to 50,000 g/mol, and 2 to 7 % sulfation (sulfate groups) per

anhydroglucose unit, which is concentrated on the surfaces of the crystals. NCC comprises crystallites whose physical dimension ranges from 5-10 nm in cross- section and 50-1000 nm in length, depending on the cellulosic material used in the extraction. These negatively charged crystallites can be dispersed in water, or other solvents, or dried to form solid powder, e.g. by freeze-drying. The NCC according to this invention comprises rod- shaped nanoparticles, derived from the crystalline regions of native cellulose.

The NCC is in native cellulose I form. In the NCC according to this invention, anionic groups are bound to the crystal surfaces either directly or via a spacer, such as a spacer comprising amine groups. For example, a suitable spacer can be created by reacting desulfated nanoparticles first with a compound containing epoxides, followed by a compound comprising amine groups. Then this compound is reacted with an appropriate reagent to substitute at least part of the hydroxyl groups with other anionic groups, preferably sulfonate, phenyl sulfate or phenyl sulfonate groups. In this way, a spacer is created between the anionic substituents and the surface of the nanocrystalline cellulose (NCC). As suitable compounds having epoxides and spacer-forming amine groups can be mentioned epichlorohydrin and 2,2'-(ethylenedioxy)bis(ethylamine) (EBEA), respectively. Surface sulfate groups can be selectively removed from NCC via alkali treatment at elevated temperatures, which provides an abundance of hydroxyl groups and low surface charge for further derivatization [Kloser, E. et al., Langmuir 2010, 26, 13450-13456]. NCC can then be derivatized with other target- specific

functionalities such as phenyl sulfonate groups by reaction of 4-sulfophenyl isothiocyanate sodium salt monohydrate (4-SPITC) with surface hydroxyl groups or amine derivatives of NCC under alkaline conditions. NCC bearing hydroxyl and/or anionic acid groups on their surfaces, i.e. sulfate, sulfonate, phenyl sulfate, or phenyl sulfonate groups, can act as an anti- viral compound alone or in another composition. It has been determined that NCC is effective in reducing the risk of infection of primate cells in vitro by Semliki Forest Virus (SFV), in particular its strain A774.

The antiviral composition comprising the NCC and a carrier can be either a pharmaceutical formulation to be administered to a mammal, or a non- pharmaceutical composition such as a disinfectant.

The pharmaceutical formulation can be, for example,

- a topical formulation, such as a cream, ointment, lotion, suspension,

emulsion, microemulsion, solution; or

- a parenteral formulation such as an intravenous, intramuscular, intranasal, intracranial, intraperitoneal, or subcutaneous formulation; or

- a solid or liquid oral formulation such as a powder, tablet, capsule,

suspension or emulsion.

The typical dose of the antiviral NCC depends on the administration route, the viral infection and the severity thereof.

The antiviral compositions can additionally comprise other active ingredients.

Next, the invention will be described more in detail by reference to the experiments. Referring to Figure 1, the experimental results indicate that the inhibition of SFV infection of primate cells in vitro is highly dependent on the concentration of NCC. In this case, at a concentration of 0.20 % wt. NCC, almost 100 % inhibition of SFV is achieved.

Referring to Figure 2, the experimental results indicate that all batches of NCC derived from cotton fiber and NCC derived from Whatman filter paper are non- cytotoxic at concentrations showing the inhibition of SFV infection because human corneal epithelial (HCE) cell viability is equal to or higher than 80 % after 1 hour of exposure in vitro.

Referring to Figure 3, the experimental results indicate that NCC derived from cotton fiber modified with phenyl sulfonate groups (NCC-SPTC) at 0,3 % wt. is effective at almost fully (> 90%) inhibiting SFV at 3,25* 10⁷ PFU/ml, while unmodified NCC derived from cotton fiber is effective at almost fully (90%) inhibiting SFV at 3,25* 10⁵ PFU/ml.

These results indicate that NCC derived from cotton fiber modified with phenyl sulfonate groups is a more effective anti-viral compound compared to NCC derived from cotton fiber bearing sulfate groups. Thus, by changing the type of anionic acid groups on the surfaces of NCC, the anti- viral activity can be fine tuned. In this case, phenyl sulfonate groups have similar chemical structure to sulfated tyrosine residues of proteins, thus are considered "tyrosine sulfate mimetic" functionalities. The results in Table 1 indicate that NCC derived from cotton fiber bearing sulfate groups (batch 1 and 2), NCC derived from cotton fiber bearing phenyl sulfonate groups (NCC-SPTC and NCC-EBEA) and NCC derived from cotton fiber after desulfation (NCC-DES) all are able to inhibit SFV infection of primate cells in vitro. The abbreviations have the following meaning: NCC-DES stands for desulfated NCC; NCC-SPTC stands for stands for NCC bearing phenyl sulfonate groups directly bound to the crystal surfaces of NCC; and NCC-EBEA stands for stands for NCC bearing phenyl sulfonate groups bound via an EBEA spacer to the crystal surfaces of NCC. EBEA stands for 2,2'-(ethylenedioxy)bis(ethylamine).

However, the experimental results indicate that the inhibition of SFV infection of primate cells in vitro is highly dependent on the concentration and surface functionality of NCC. In the case of NCC bearing sulfate groups, almost 100 % inhibition is achieved at 0.2 % wt. NCC. In the case of NCC bearing phenyl sulfonate groups (NCC-SPTC and NCC-EBEA), almost 100 % inhibition is achieved at 0.1 % wt. These results indicate that NCC bearing phenyl sulfonate groups are more effective at inhibiting SFV in vitro. Thus, the anti-viral activity of NCC can be fine tuned with different anionic acid groups and/or hydroxyl groups.

Referring to Figure 4, the experimental results indicate that NCC derived from cotton fiber modified with phenyl sulfonate groups (NCC-SPTC) and (NCC-EBEA) are non-cytotoxic at concentrations showing the inhibition of SFV infection because human corneal epithelial (HCE) cell viability is equal to or higher than 80 % after 1 hour of exposure in vitro.

Referring to Figure 5, the experimental results indicate that NCC derived from cotton fiber after desulfation (NCC-DES) are non-cytotoxic at 0,1 % wt. because human corneal epithelial (HCE) cell viability is equal to or higher than 80 % after 1 hour of exposure in vitro. These results also indicate that NCC modified with anionic acid groups (Cotton NCC, Whatman NCC, NCC-SPTC, NCC-EBEA) is less cytotoxic to HCE cells at 0,3 and 0,4 % wt than NCC after desulfation (see Figure 2 and 4).

Table 1 summarizes the influence of NCC derived from cotton fiber bearing sulfate groups (batch 1 and 2), NCC derived from cotton fiber bearing phenyl sulfonate groups (NCC-SPTC and NCC-EBEA) and NCC derived from cotton fiber after desulfation (NCC-DES) as a function of concentration on the inhibition of SFV infection in vitro. Samples containing only buffer and viral vector were used as a control.

Referring to Table 1, the experimental results indicate that the inhibition of SFV infection of primate cells in vitro is highly dependent on the concentration and surface functionality of NCC. In the case of NCC bearing sulfate groups, almost 100 % inhibition is achieved at 0,2 % wt. NCC. In the case of NCC bearing phenyl sulfonate groups (NCC-SPTC and NCC-EBEA), almost 100 % inhibition is achieved at 0,1 % wt. These results indicate that NCC bearing phenyl sulfonate groups are more effective at inhibiting SFV in vitro. In the case of NCC derived from cotton fiber after desulfation (NCC-DES), 100 % inhibition was achieved with 0,36 % wt. Thus, the anti- viral activity of NCC can be fine tuned with different anionic acid groups and/or hydroxyl groups.

Preferred embodiments are described in more detail by the following non-limiting examples:

Example 1. Preparation of nanocrystalline cellulose (NCC) bearing sulfate groups

NCC is obtained from Whatman 1 filter paper or cotton fibers by acid hydrolysis with 65 wt% aqueous sulfuric acid solution (45 °C, 45 minutes). The resulting dispersion is diluted and filtered into ice cubes and washed with distilled water until neutral pH by successive centrifugations at 12,000 rpm at 4 °C for 20 minutes. Finally, dialysis for one week against distilled water with a 12,400 MWCO dialysis membrane is performed to remove trace amounts of residual sulfuric acid.

Concentrations of the resulting NCC dispersions are calculated gravimetrically.

Example 2. Desulfation of nanocrystalline cellulose (NCC)

Removal of sulfate groups derived from sulfuric acid hydrolysis of NCC is necessary to obtain the maximum number of hydroxyl groups. Aqueous dispersions of NCC are treated in 1 M NaOH at 60 °C for 5 hours. Subsequently, the reaction is quenched by a 10-fold dilution with distilled water, centrifuged at 12,000 rpm at 4 °C for 20 minutes, re-dispersed and dialyzed against distilled water for one week to remove traces of base. These samples are referred to as NCC-DES.

Example 3. Preparation of nanocrystalline cellulose (NCC) bearing phenyl sulfonate groups

After the desulfation reaction (see Example 2), 4-sulfophenyl isothiocyanate sodium salt monohydrate (4-SPITC) is added and allowed to react at 40 °C for 20 hours. Subsequently, the reaction is quenched by a 10-fold dilution with distilled water, centrifuged at 12,000 rpm at 4 °C for 20 minutes, re-dispersed and dialyzed against distilled water for one week to remove residual reactants and traces of base. These samples are referred to as NCC-SPTC.

Example 4. Preparation of nanocrystalline cellulose (NCC) bearing phenyl sulfonate groups with molecular spacers in aqueous media

Epichlorohydrin is added directly after the desulfation reaction (see Example 2) and stirred at 40 °C for 20 hours. Then, the reaction is diluted, centrifuged and re- dispersed, then brought to pH = 11 with a few drops of 1 M NaOH. Subsequently, 2,2'-(ethylenedioxy)bis(ethylamine) (EBEA) is added and reacted at 40 °C for 20 hours. The reaction is then dialyzed for 24 hours and brought to pH = 11. Then, 4- sulfophenyl isothiocyanate sodium salt monohydrate (4-SPITC) is added and reacted at 40 °C for another 20 hours. The products are then collected by centrifugation, re-dispersed and dialyzed for one week. These samples are referred to as NCC-EBEA.

Example 5. Preparation of nanocrystalline cellulose (NCC) bearing phenyl sulfonate groups with molecular spacers in organic media

After the desulfation reaction (see Example 2), NCC is solvent-exchanged from water to acetone to anhydrous dimethyl sulfoxide (DMSO) by successive centrifugations at 12,000 rpm at 4 °C for 20 minutes. Under nitrogen atmosphere, 1,1 '-carbonyldiimidazole (CDI) is added to the reaction mixture and stirred at 40 °C for 20 hours. Then, the reaction is diluted, centrifuged and re-dispersed in DMSO. Subsequently, 2,2'-(ethylenedioxy)bis(ethylamine) (EBEA) is added and reacted at 40 °C for 20 hours. Again, the reaction is diluted, centrifuged and re-dispersed in DMSO. Then, 4-sulfophenyl isothiocyanate sodium salt monohydrate (4-SPITC) is added and reacted at 40 °C for another 20 hours. The products are then collected by centrifugation, re-dispersed in water and dialyzed for one week. Example 6. Sample buffering and anti- viral activity of NCC

Samples are buffered with 50 mM Tris-HCl. Final buffer concentration is 10 mM for Whatman NCC samples and 50 mM for Cotton NCC samples. pH is adjusted to 7.8. Samples diluted with 10 mM Tris-HCl buffer.

NCC and Semliki Forest virus (SFV) vector VA7-EGFP stock are mixed together, for example 100 μl both, so final volume is 200 μl. NCC concentration and virus concentration both drop to half because of dilution (for example NCC derived from cotton fiber 0,73%→0,365%). Solutions are incubated in refrigerator overnight and next day the number of infectious virus particles is measured. Antiviral activity will be seen as a loss of infectious particles. Number of infectious particles is measured by plaque titer protocol. Number of infectious virus particles is related to inhibition (i.e. 100% inhibition represents complete loss of viral activity).

Example 7. Effect of NCC on cell viability (cytotoxicity)

To ensure that the antiviral activity of nanocrystalline cellulose (NCC) samples is not due to cytotoxicity, Vero (B) cells or human corneal epithelial (HCE) cells are treated with the NCC samples for 1 hour. Cells are washed with phosphate buffer solution and death of cells is quantified with MTT assay (colorimetric assay), wherein at least 80 % cell viability indicates non-cytotoxicity.

Industrial Applicability

The antiviral fabric, useful for disinfection purposes, can be used, for example, in masks, air cleaning filters, gloves, sheets, curtains, wears and protective cloths, and the like. Further contemplated applications include uses in furniture, door handles, sanity rooms and various surfaces and equipment which are being manipulated and touched by a plurality of users. NCC can be used as a disinfectant. Citation List

Patent Literature WO 9712621

WO 2002002189

WO 2012068670

US 8309708 B2

US 2007014812 A1

US 20090247740 A1

JP 2008266265A

EP 2040716 B1

US 7235536 B2

WO 2005004882 A1

Non Patent Literature

Teixeira, C. et al, Eur. J. Med. Chem. 2011, 46, 979-992.

Pirrone, V. et al., Antiviral Res. 2011, 90, 168-182.

Flexner, C. et al., Antimicrob Agents Chemother 1991, 35, 2544-50.

Van Damme, L. et al., N. Engl. J. Med. 2008, 359, 463-472.

Stone, M. J. et al., New Biotechnol. 2009, 25, 299.

Di Gianvincenzo, P. et al., Bioorg. Med. Chem. Lett. 2010, 20, 2718.

Acharya, P. et al., ACS Chem. Biol. 2011, 6, 1069-1077.

Rowe, R. C. et al. (editors), Handbook of Pharmaceutical Excipients,

Pharmaceutical Press and American Pharmacists Association, 2012.

O'Connor, R. E. et al., Pharm. Res. 1993, 10, 356-361.

FDA SCOGS database; Report No. 25, 1973; Accessed February 5, 2013.

Lam, E. et al., Trends Biotechnol. 2012, 30, 283-290.

Jackson, J. K. et al., Int. J. Nanomed. 2011, 6, 321-330.

Dong, S. et al., Nano LIFE 2012, 2, 1241006. Viiha-Koskela MJ, Tuittila MT, Nygardas PT, Nyman JK, Ehrengruber MU, Renggli M, Hinkkanen AE. 2003. J. Neurovirol. 9: 1-15.

Claims

1. Nanocrystalline cellulose (NCC) as an antiviral compound.

2. The antiviral compound according to claim 1, characterized in that said NCC bears hydroxyl groups and/or anionic groups on the crystal surfaces.

3. The antiviral compound according to claim 1 or 2, characterized in that said anionic groups comprise sulfate, sulfonate, phenyl sulfate or phenyl sulfonate groups.

4. The antiviral compound according to claim 3, characterized in that said anionic groups are bound to the crystal surfaces either directly or via a spacer, such as a spacer comprising amine groups.

5. The antiviral compound according to any of the preceding claims, characterized in that said NCC comprises rod-shaped nanoparticles, derived from the crystalline regions of native cellulose.

6. The antiviral compound according to any of the preceding claims, characterized in that said NCC is in native cellulose I form.

7. The antiviral compound according to any of the preceding claims, characterized in that said NCC has a molecular weight in the region 15000 to 50000 g/mol.

8. The antiviral compound according to any of the preceding claims, characterized in that said NCC has a particle size in the range 5 nm to 1000 nm, where the particle size is defined as the dimension corresponding to the diameter of a sphere encasing the nanoparticle.

9. The antiviral compound according to any of the preceding claims, characterized in that said NCC is derived as rod-shaped nanoparticles from crystalline regions of native cellulose by treating said native cellulose with strong sulfuric acid, wherein the rod-shaped nanoparticles are acid-insoluble and have a degree of sulfation from 2 to 7 %.

10. A method for the preparation of a nanocrystalline cellulose (NCC) useful as an antiviral compound according to any of the claims 1-9, characterized by

(a) treating native cellulose with a strong acid, preferably strong sulfuric acid, most preferably a 65 wt-% aqueous sulfuric acid solution, to dissolve the amorphous regions of the cellulose,

(b) separating the acid-insoluble rod shaped, sulfated nanoparticles from the dissolved amorphous cellulose,

(c) recovering the acid-insoluble rod shaped, sulfated nanoparticles, and optionally

(d) desulfating said sulfated nanoparticles, and optionally further substituting hydroxyl groups on the desulfated nanoparticles with other anionic groups.

11. The method according to claim 10, characterized in that the acid-insoluble rod shaped, sulfated nanoparticles recovered in step (c) in claim 10 are treated with a base, preferably NaOH, to give desulfated nanoparticles, where sulfate groups are replaced with hydroxyl groups.

12. The method according to claim 10 or 11, characterized in that the desulfated nanoparticles are reacted with appropriate reagents to substitute at least part of the hydroxyl groups with other anionic groups, preferably sulfonate, phenyl sulfate or phenyl sulfonate groups.

13. The method according to any of the claims 10-12, characterized in that the desulfated nanoparticles are reacted with a reagent suitable for substituting hydroxyl groups with phenyl sulfonate groups, for example with 4-sulfophenyl

isothiocyanate or its salt, to give nanocrystalline cellulose (NCC) with phenyl sulfonate substituents.

14. The method according to any of the claims 10-13, characterized in that the desulfated nanoparticles are first reacted with a compound comprising epoxide groups, followed by amine groups and then reacted with an appropriate reagent to substitute at least part of the hydroxyl groups with other anionic groups, preferably sulfonate, phenyl sulfate or phenyl sulfonate groups, wherein a spacer is created between the anionic substituents and the surface of the nanocrystalline cellulose (NCC).

15. The method according to any of the claims 10-14, characterized in that the epoxide group comprising compound and spacer-forming amine groups comprising compound is epichlorohydrin and 2,2'-(ethylenedioxy)bis(ethylamine) (EBEA), respectively.

16. An antiviral composition comprising an antiviral compound and a carrier, characterized in that the antiviral compound is a nanocrystalline cellulose (NCC) according to any of the claims 1-9.

17. The antiviral composition according to claim 16, characterized in that the composition is a pharmaceutical formulation for treatment or prevention of viral infections.

18. The antiviral composition according to claim 16 or 17, characterized in that the pharmaceutical formulation is

- a topical formulation, such as a cream, ointment, lotion, suspension,

emulsion, microemulsion, solution; or

- a solid or liquid oral formulation such as a powder, tablet, capsule,

suspension or emulsion.

19. The antiviral composition according to any of the claims 16-18, characterized in that the composition additionally comprises other active ingredients.

20. An antiviral fabric useful for disinfection purposes, especially for reducing the risk of viral infections in hospitals, clinics and the like, characterized in that said fabric is a woven or non-woven fabric immersed with an antiviral nanocrystalline cellulose (NCC) according to any of the claims 1-9 or an antiviral composition comprising antiviral nanocrystalline cellulose (NCC) and a carrier according to claim 16.

21. The use of the antiviral fabric according to claim 20 in masks, air cleaning filters, gloves, sheets, curtains, wears and protective cloths, and the like.

22. The use of the antiviral fabric according to claim 20 in furniture, door handles, sanity rooms and for forming surfaces and equipment which are being manipulated and touched by a plurality of users.

23. The use of nanocrystalline cellulose (NCC) as a disinfectant.

24. A method for preventing or treating viral infections in a mammal, said method comprising administering to said mammal in the need thereof an effective amount of an antiviral nanocrystalline cellulose (NCC) according to any of the claims 1-9.