FIELD OF THE INVENTION

The present invention relates to one or more non-surfactant compounds of biodegradable polymers comprising one or more modified polysaccharide(s) for immobilization of viral particles and/or inhibition of the virus. The invention further relates to PPE materials and articles treated with at least one nonsurfactant compound of the invention, and to a method of immobilization of viral particles and/or inhibition of the virus with the use of a PPE material comprising at least one non-surfactant compound of the invention.

BACKGROUND OF THE INVENTION

The outbreak of novel coronavirus (SARS-CoV-2) infections globally has placed an unprecedented burden on all facets of our modern society. Restrictions on almost all physical interactions between people have been imposed across the entire globe, impacting on freedom of movement, gatherings, socialization, commerce, schooling and more. An unprecedented mustering of efforts and resources probably never seen since World War II is taking place to contain the disease, which was officially named Covid-19 by the World Health Organization (WHO). Leading the fight against the disease are healthcare workers and professionals ranging from diagnostic technicians and nurses to doctors. These workers are provided with personal protective equipment (PPE) that may be just a basic facemask and gloves or a full hazmat suit with a respirator, depending on the risk of exposure, the level of contamination they would be encountering, and/or on the resources available to healthcare workers.

Furthermore, in most countries around the globe it is acknowledged that the wearing of a facemask of some sort will only assist in curbing the spread of the virus if the facemask is worn by both the infected person and the uninfected person.

Masks offer a general and non-specific physical barrier against any particulate matter. However, it has been reported that the most commonly available surgical facemask offers little to no protection against viral infection of the wearer via the nose and mouth, which are the most important routes for pulmonary infection by respiratory viruses, unless the infected person is also wearing a facemask (Makison Booth et al, 2013). Sadly, particularly in resourcepoor settings, this is often the only PPE available.

In South Africa, the Department of Health has expressed serious concerns at the high rate of infections among healthcare workers. While not all of these cases of infections may be directly attributable to the inadequacies of the facemask or other PPE available, the risk of exposure due to these inadequacies cannot be ignored.

One major risk factor for healthcare workers in particular, is that the very nature of their duties exposes them on a regular and even continuous basis to seriously ill patients or contaminated body fluid samples. The healthcare worker is therefore continuously exposed to the virus. The loss of a single health worker has severe reverberations beyond the immediate Covid-19 crisis. For every single health worker lost, potentially hundreds of ordinary citizens will be vulnerable to Covid-19 and other diseases. However, other service providers in essential services including cashiers, those in the hospitality industry, emergency service personnel, teachers, and the like can also be exposed to virus for extended periods of time, often in settings where there is poor ventilation and social distancing is difficult or not adhered to and where there are many people not wearing masks at all. Unfortunately, the wearing of a surgical mask or cloth mask is not effective protection in such a setting.

Various materials have been used to produce masks. N95 masks are believed to offer the best protection against transmission of coronavirus particles. Although not specifically designed to protect against viruses they are able to prevent passage of at least 95% of particles with size greater than 300 nm. During the current Covid-19 pandemic, these have globally become very scarce and relatively expensive. This has limited their widespread use, even in clinical settings. Also, because of the multiple layers of tightly interwoven fibers used to make N95 masks, breathing discomfort is often experienced.

More widely available are surgical masks. These are more breathable but this comes at the expense of a lower ability to prevent virus transmission. Currently, they cost as low as just a quarter of the price of an N95 mask.

However, use of at least cloth masks by the general public has also been recommended, as these are readily obtainable, and are washable and reusable. However, ordinary cloth fabrics offer limited and variable protection against viral transmission. As South Africa and many countries make efforts to return to a semblance of normality, it is universally accepted that normalcy will, at least for the short to medium term, be centred around ensuring that infections from SARS-CoV-2 be kept under controllable limits. Wearing of masks should be required at every mass gathering, including at schools, sporting events, work places, on public transport, in shopping centres and the like. Enhancing the protectiveness of surgical and cloth masks may prove to be the trump card in winning the battle of controlling the transmission of the coronavirus and saving lives. Compounds and compositions that may be used to treat or that may be incorporated into the material of such masks during manufacture of the material and that can significantly enhance the inhibition of the movement of viral particles through the mask material or inactivate the virus are urgently needed. Preferably such materials should not add significantly to the cost of the material or mask, and should still provide for a breathable mask. Such improved masks may be useful not only against the current Covid-19 pandemic, but against other viruses as well.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided one or more nonsurfactant biodegradable polymer compound(s) comprising one or more modified polysaccharide(s) for immobilization of viral particles and/or inhibition of a virus wherein the modified polysaccharide(s) are hydrophobically modified polysaccharide(s) and/or are modified to have a cationic charge. The one or more modified polysaccharide(s) may be selected from the group comprising or consisting of chitosan, starch, cellulose and alginate.

In particular, the modified polysaccharide(s) may be chemically modified with one or more acyl, alkyl and/or aryl groups to have a cationic charge and/or be chemically modified with one or more hydrophobic substituents, including long chain fatty acyl chlorides and/or long chain fatty acyl N-hydroxysuccinimide (NHS) esters or by akylation with long aliphatic chains to have amphiphilicity.

For example the cationically charged modified polysaccharide(s), preferably chitosan, may comprise multiple substitutions of an amino group resident on the polysaccharide(s), or may comprise a quaternized amino substituent on the polysaccharide(s). In particular, the cationically charged modified polysaccharide(s), preferably chitosan, may comprise a tri-substitution of the primary amine of an amine polysaccharide such as chitosan, or an amine-functionalised polysaccharide such as alginate amine, with one or more alkyl and/or aryl group(s).

Alternatively, or in addition, the cationically charged modified polysaccharide(s), preferably chitosan, may comprise any one of a grafted or protonated cationic amino, imino, ammonium, sulfonium, or phosphonium group on the primary amine of an amine polysaccharide such as chitosan, or an amine-functionalised polysaccharide such as alginate amine, or on one or more hydroxyl group(s) of any polysaccharide(s). In particular, the ammonium group may be glycidyltrimethylammonium, which when reacted with chitosan forms N-(2- hydroxy)propyl-3-trimethyl ammonium chitosan chloride (HTCC-CS) or trimethylallyl ammonium.

For example, the amphiphilic hydrophobically modified polysaccharide(s) may comprise a portion of hydroxyl and/or amino groups functionalised with hydrophobic substituents, such that the remaining free -OH and/or NH2 groups are available to form hydrogen bonding with water molecules despite having hydrophobic chains. In particular, the amphiphilic hydrophobically modified polysaccharide(s) may comprise-OH and/or -NH2 groups functionalised with long chain fatty acyl chlorides and/or long chain fatty acyl N-hydroxysuccinimide (NHS) esters or by akylation with long aliphatic chains to have amphiphilicity.

In particular, the modified polysaccharides may comprise any one or more of the modified polysaccharides as set out in Table 1 , or may be selected from the group consisting of any one or more of the modified polysaccharides as set out in Table 1.

Further in particular, the modified polysaccharides may be any one or more of hydroxylpropanoic acid-chitosan salt (HPA/CS), N-(2-hydroxy)propyl-3- trimethyl ammonium chitosan chloride (HTCC-CS), hexadecyl (HexD-CS), lauroyl- or stearoyl-chitosan. According to a further aspect of the invention, there is provided a method of manufacturing a non-surfactant biodegradable polymer compound comprising one or more modified polysaccharide(s) for immobilization of viral particles and/or inhibition of a virus, wherein the modified polysaccharide(s) have a cationic charge and/or are amphiphilic polysaccharide(s), the method comprising a step of modifying the one or more polysaccharide(s):

(a) to have a cationic charge by introduction of one or more acyl, alkyl and/or aryl groups or by introduction of a quaternized amino substituent into the one or more polysaccharide(s), and/or

(b) to have amphiphilicity by chemical modification of the one or more polysaccharide(s) with one or more hydrophobic substituents, including long chain fatty acyl chlorides and/or long chain fatty acyl N-hydroxysuccinimide (NHS) esters or by akylation with long aliphatic chains to have amphiphilicity.

The one or more modified polysaccharide(s) are preferably selected from the group comprising or consisting of chitosan, starch, cellulose and alginate.

In particular, the cationic chemical modification may be by tri-substitution of alkyl and/or aryl groups of the primary amine of chitosan or an amine-functionalised polysaccharide such as alginate amine; or by grafting or protonation of any one of cationic amino, imino, ammonium, sulfonium, or phosphonium groups on the primary amine of chitosan or an amine-functionalised polysaccharide such as alginate amine or the hydroxyl groups of any polysaccharide(s). In particular, the ammonium group may be glycidyltrimethylammonium or trimethylallyl ammonium. When the ammonium group is glycidyltrimethylammonium, the reacted chitosan product is N-(2-hydroxy)propyl-3-trimethyl ammonium chitosan chloride (HTCC-CS).

To confer amphiphilicity, the chemical modification may be by functionalising a portion of the hydroxyl and/or amino groups of any polysaccharide(s) with hydrophobic substituents, such that the remaining free -OH and/or NH2 groups are available to form hydrogen bonding with water molecules despite having hydrophobic chains. In particular, the functionalising is by reaction of the -OH and/or -NH2 groups with long chain fatty acyl chlorides and/or long chain fatty acyl N-hydroxysuccinimide (NHS) esters after activation with an appropriate carbodiimide reagent, including an alkyl halogen such as an alkyl bromide, alkyl chloride or alkyl iodide. Alternatively, the functionalising is by akylation with long aliphatic chains.

Hydrophobic modification proceeds through a nucleophilic attack of a lone pair of electrons on the -OH or -NH2 of the polysaccharide on the activated long chain acyl or alkyl substituent.

According to a further aspect of the invention there is provided method for producing or treating a PPE article or material for immobilization of viral particles and/or inhibition of a virus, comprising the use of a non-surfactant biodegradable polymer compound comprising one or more cationically and/or hydrophobically modified polysaccharide(s) of the invention as described above.

The non-surfactant biodegradable polymer compound comprising one or more cationically and/or hydrophobically modified polysaccharide(s) may be incorporated into an existing PPE article by treatment of the PPE article with the non-surfactant biodegradable polymer compound. Alternatively, the nonsurfactant biodegradable polymer compound comprising one or more cationically and/or hydrophobically modified polysaccharide(s) may be used to treat the material used to produce PPE articles prior to or during the process of production of the PPE articles.

For example, the non-surfactant biodegradable polymer compound comprising one or more cationically and/or hydrophobically modified polysaccharide(s) may be dispursed into a suitable solvent including water and/or alcohol and stirred to form a uniform solution prior to spraying or immersing the PPE material or PPE article in the solution. For example, the PPE material or article may be comprised of a cloth fabric or a non- woven fabric. The non-woven fabric may be a polypropylene, polystyrene, polycarbonate, polyethylene, or polyester non-woven fabric. Preferably, the non-surfactant biodegradable polymer compound is cationically and/or hydrophobically modified chitosan. Further preferably, the non-woven fabric is polypropylene (PP).

The method may further comprise a step of drying the fabric. Drying may be at room temperature.

According to a further aspect of the invention, there is provided a PPE material or article for for immobilization of viral particles and/or inhibition of a virus treated with or comprising a non-surfactant biodegradable polymer compound comprising one or more cationically and/or hydrophobically modified polysaccharide(s) of the invention, or produced according to the above method of the invention.

For example, the PPE material may be a cloth fabric or a non-woven fabric. The non-woven fabric may be a polypropylene, polystyrene, polycarbonate, polyethylene, or polyester non-woven fabric.

For example, the PPE article may be a cloth mask or a surgical mask. The PPE may be disposable.

According to a further embodiment of the invention, there is provided a method of immobilization of viral particles and/or inhibition of a virus comprising use of the non-surfactant biodegradable polymer compound comprising one or more cationically and/or hydrophobically modified polysaccharide(s) of the invention as described above, or of a PPE material or article of the invention as described above.

The virus may be an enveloped virus. The virus may be a virus causing a respiratory tract and/or pulmonary infection. The virus may be a virus that is transmitted to a subject through inhalation via the nose and/or mouth. For example, the virus may be an influenza virus, a rhinovirus, a coronavirus including SARS-CoV, SARS-CoV-2, or MERS-CoV, respiratory syncytial viruses, human metapneumovirus, and parainfluenza viruses.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows A. A schematic of packing of test samples in syringe for antiviral activity testing by filtration (Hm = hydrophobically modified), and B. A schematic of pseudo-HIV particles filtration through PPE fabric impregnated with antiviral polysaccharide material;

Figure 2 shows membrane disruption experiments by haemolysis with modified chitosan. Graph showing the concentration-dependent haemolytic effect of modified chitosan materials. Extensive lysis resulted in release of haemoglobin into solution in the experimentals while unlysed cells are pelleted in the negative control. Polyethylenimine (PEI) is a synthetic polycationic polymer used as control.

Figure 3 shows A. antiviral activity of a polypropylene-wool (PP-wool) fabric coated with HM-Chitosan (B2), Quaternary-CS (C2; Synthetic Method 07), HPA/CS (D2; Synthetic Method 08), a 50:50 mixture of B2 and C2 (A2) and uncoated PP-wool (02). B. Reference experiments with N95 respirator material (07) and 0.4 pm filter membrane (00) used to line the bottom of the syringes. The biological controls are: VC = virus control (i.e. not filtered through any material), CC = mammalian cell control (i.e. not infected with virus); and

Figure 4 shows antiviral activity of cationic chitosan material HPA/CS (A1 ) and quaternary chitosan HTCC-CS (C1 ) coated onto polypropylene. The viral control (VC) is uninhibited SARS-CoV-2 virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to one or more one or more non-surfactant biodegradable polymer compound(s) comprising one or more modified polysaccharide(s) for immobilization of viral particles and/or inhibition of a virus, wherein the modified polysaccharide(s) are hydrophobically modified polysaccharide(s) and/or are modified to have a cationic charge. The invention further relates to PPE materials and articles treated with the at least one modified non-surfactant biodegradable polymer compound(s) of the invention, and to a method of immobilization of viral particles and/or inhibition of the virus with the use of a PPE material comprising the one or more modified nonsurfactant biodegradable polymer compound(s).

The applicant has discovered that cationically or hydrophobically modified polysaccharides or polysaccharides having both cationic and hydrophobic modification, such as chitosan, starch, cellulose, alginate etc. may be successfully used as or comprised in non-surfactant biodegradable polymer materials for the immobilization of enveloped viral particles and inhibition of enveloped viruses by intercalating into the virus lipid envelope.

The cationic charged molecules are able to initiate electrostatic immobilization of the virus because the viral envelope has a counter anionic charge from phosphate groups. The long-chain amphiphilic molecules are able to disrupt the envelope of the virus by insertion of long hydrophobic chains into the envelope, while the hydrophilic head interacts with an aqueous phase to result in tiny water-soluble micelles. When incorporated together in a non-surfactant biodegradable polymer of the invention and applied to PPE material or a PPE article, the cationic and long-chain hydrophobic modifications act synergistically to improve the immobilization of viral particles and/or inhibition of the virus. In one embodiment of the invention, the applicant has grafted long hydrophobic chains including long chain fatty acyl chlorides and/or long chain fatty acyl N- hydroxysuccinimide (NHS) esters onto hydrophilic polymers such as chitosan, starch, cellulose and alginate to form amphiphilic polymers that mimic the amphiphilicity of detergents.

In an alternative embodiment of the invention, the applicant has added a permanent cationic charge on the polysaccharide(s) to attract the anionic envelope of the virus.

Where the two embodiments are combined, this further strengthens the electrostatic attractiveness of the polymeric materials and virus capture, virus immobilization and virus inactivation properties. The cationic charge can be as a result of multiple acyl, alkyl and/or aryl group or quaternized amino substituent substitutions on an amino group resident on the polysaccharide(s), used together with the long chain fatty acyl chlorides and/or long chain fatty acyl N- hydroxysuccinimide (NHS) esters grafted to the hydroxyl and/or amino groups of the polysaccharide(s).

These modified non-surfactant biodegradable polymer compound(s) can either be readily incorporated into existing PPE materials or be processed into entirely new PPE materials.

The modified non-surfactant biodegradable polymer compound(s) can be used to treat cloth fabric and other non-medical masks as well as surgical masks, including by spraying or immersion. By incorporating the modified nonsurfactant biodegradable polymer compound(s) of the invention into a cloth fabric or non-woven fabric, the immobilization of viral particles and/or inhibition of the virus by the mask can be significantly enhanced. The applicant’s invention will improve the antiviral ability of the common surgical mask to a performance level comparable to N95 without significantly affecting cost and breathability.

In one embodiment, the modified non-surfactant biodegradable polymer compound(s) are formulated for application onto PPE such as by spraying or immersion after manufacture and prior to use. It is expected that PPE so treated with the formulation will lose its antiviral property during normal washing process. Hence, this approach is suggested for disposable PPE.

According to a second embodiment of the invention, the hydrophobically modified polymer is incorporated during the manufacturing process of fabric materials including cloth before the final PPE is produced.

The key benefit of this technology is that it will provide PPE with antiviral activity that is not based on metallic ions or merely passive filtration of viral particles as a result of multiple fabric layers. As the world battles to enter a post-COVID world, it is becoming clear that an inflection point might have been passed for medical grade PPE.

The modified polymers of the invention have been successfully developed and inhibition activity was observed in a biological inhibition assay in vitro using an HIV pseudo-virus.

The exemplary examples below are for illustrative purposes and should in no way be construed as limiting in any way the scope of the invention.

EXAMPLES

1. Background

Polysaccharides are the most abundant naturally occurring polymers. With an abundance of nucleophilic hydroxyl and amino groups, they lend themselves to a wide range of functional group modifications. By substituting on the polymeric backbone, desired properties and functionalities like antimicrobial activity can be conferred on to the otherwise inactive polymer. Polysaccharides are also attractive as platform polymers for material development because of their biodegradability in spite of their very good mechanical properties.

Described below is a wide, but certainly not exhaustive, selection of chemical modification techniques to introduce long chain fatty acids and cationic groups onto chitosan, cellulose and starch to confer them with antiviral activity. These polysaccharides and the chemistries of modification were selected for their enormous natural abundance and for their facility, respectively.

Fatty acid chains were conjugated to the hydroxyl and amino groups via ester and amide linkages. The carboxylic groups were activated by conversion to acid chlorides or by carbodiimide chemistry. Alkylation to form ethers and amines was accomplished with alkyl bromides. Cationic charges were conferred by either carrying out trialkylation of the primary amine of chitosan or by grafting trialkylammonium substituents onto the polymer.

2. Methods and Materials

2.1. Materials

Chitosan of at least 90% deacetylation was purchased from DB Fine Chemicals Pty (Ltd), South Africa. All other reagents and solvents were purchased from Sigma-Aldrich, South Africa, and used as received. Stearoyl chloride was prepared in our laboratory and stored at -20 °C until used.

2.2. Methods

Hydrophobic Modification of Polysaccharides by Acylation with Long Chain Fatty Acids

Method 01. To a stirred homogeneous suspension of the polysaccharide chitosan (1 g, 6.21 mmol, 1 mol eqv) in anhydrous dimethylformamide (DMF) (10 ml), was added lauroyl chloride dropwise. The reaction solution was alkalized with triethylamine and left to react under vacuum at 25 °C for 18 h. At the end of the reaction period, the materials were washed with acetone, filtered through 0.45 pm filter paper and air-dried.

Method 02. Chitosan (2.0 g) was soaked in a mixed solvent of trimethylamine (50 ml) and acetone (30 ml) for 16 h at 50 °C, then cooled and stirred for 2 h at 0-5 °C. Lauroyl chloride (10 g), which was calculated by the mole ratio of lauroyl chloride to the reactive groups (-NH2 and -OH) of chitosan, was dissolved in anhydrous acetone (20 ml) and added dropwise into the chitosan suspension over 3 h. The temperature of the reaction solution was raised to 80 °C over 4 h and refluxed at this temperature for 3 h. To work up the reaction, the reaction mixture was filtered and washed with acetone for thrice and poured into of methanol (200 ml). The precipitate was dissolved in acetone and precipitated with methanol thrice and then dried under vacuum at 30 °C overnight to obtain the acylated chitosan.

For acylation of cellulose and starch, the same procedure was followed except for a variation in solvent systems. The typical solvent systems used include triethylamine/ dimethylsulphoxide, triethylamine/ dimethylformamide, and triethylamine/ acetone.

Typical long-chain fatty acid chain lengths can be from C-6 to C-18.

Method 03. The polysaccharide chitosan (2.0 g, 1 1.2 mmol, 1.0 eqv) was completely dissolved in aqueous acetic acid solution (1 % v/v, 100 ml) and lyophilized. The dried chitosan was then re-dissolved in dH2O (200 ml) and DMF (40 ml) was added to the solution slowly without precipitating the polymer. To a separate stirring solution of the fatty acid lauric acid (120 mg, 0.56 mmol, 0.05 eqv) in DMF (10 ml) was added N-Ethyl-N'-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC HCI) (233 mg, 1.4 mmol, 0.15 eqv) and N-hydroxysuccinimide (NHS) (207 mg, 1 .8 mmol, 0.15 eqv) and the reaction was left to run for 2 hours to produce NHS ester of lauric acid. The lauroyl NHS ester solution was added dropwise to the stirring binary aqueous DMF solution of chitosan and the reaction was left to run for 24 hours to yield chitosan conjugated with lauric acid. The lauric acid-conjugated chitosan was precipitated with cold ethanol and centrifuged at 6000 rpm for 20 min, twice. After the final wash, residual solvent was removed in vacuo to obtain the pure lauroyl-chitosan.

Hydrophobic Modification of Polysaccharides by Akylation with Long Aliphatic Chains

Method 04. In a typical synthesis, the polysaccharide chitosan (2 g) was alkalized at room temperature for 80 min using of sodium hydroxide (8 g) in isopropanol (40 ml). After the alkalization process, the temperature of the mixture was raised to a range of 45- 85 °C followed by the addition of 1 - bromoalkane (6 ml) drop-wise to the mixture with stirring. Stirring was continued at the same temperature for 3-5 h. To work up the process, the reaction solution was cooled and 15% HCI was added until the solution was neutralised. Then acetone was added to precipitate alkylated chitosan, which was filtered and washed with 85% methanol. The product (HexD-CS) was then dried.

For alkylation of cellulose and starch, the same procedure was followed except for a variation in solvent systems. Alkalization with sodium hydroxide was done in dimethylsulphoxide instead of isopropanol. The typical solvent systems used include triethylamine/ dimethylsulphoxide, triethylamine/ dimethylformamide, and triethylamine/ acetone.

Typical long-chain bromoalkane chain lengths can be from C-6 to C-18.

Cationic Modification of Polysaccharides

Method 05. To a stirred solution of the polysaccharide chitosan (2 g) in N- methylpyrrolidone (80 ml) was added sodium iodide (4.8 g), aqueous sodium hydroxide solution (11 ml, 15% w/v) and methyl iodide (11.5 ml). This mixture was stirred for 45 min at a temperature of 60 °C. The process was repeated three times after finishing the work up. The product was precipitated from solution with ethanol and isolated by centrifugation. The final product was dried in a vacuum oven at 40 °C.

Cationic Modification of Polysaccharides with Quaternary Alkyl Ammonium Groups

Method 06. The polysaccharide chitosan (2.0 g) was dissolved in aqueous acetic acid solution (2 wt%) in a three-neck flask equipped with a stirring apparatus and a reflux condenser. The mixture was stirred to completely dissolve the chitosan under inert nitrogen atmosphere and heated in a water bath at 60 °C for 30 min. Potassium persulfate (0.061 g) and NaHSOs (0.024 g) were added to the chitosan solution to initiate the graft polymerization. After 30 min, a 15 wt% aqueous triethylammonium allyl bromide (0.50 g) was then added to the reaction mixture. The reaction was allowed to continue for 3 h. After completion of the reaction, the solution was cooled to room temperature and the required volume of ethanol was added to precipitate the polymer. The mass was filtered and washed several times with ethanol to obtain the pure modified polymer, which was dried at 60 °C.

Method 07. Chitosan (0.3 g) was suspended in double distilled water (20 ml) and the suspension was stirred for 2 h at room temperature. Glycidyltrimethylammonium chloride (GTMAC) (3 equiv) was then added to the polymer solution in three portions at approximately 3 h intervals. The reaction temperature was raised to 85 °C for 24 h after the final addition of GTMAC. The chitosan derivative (HTCC-CS) was precipitated in cold acetone and filtered through a sintered glass funnel and washed with acetone and ethanol copiously.

Method 08. Mixed chitosan (0.5 g) was dissolved in a stirring solution of [3- hydroxypropanoic acid (HPA, 1 % v/v, 20 ml) at room temperature for 5 h to make a 25 mg/ml solution of HPA/CS. For antiviral assay, 50 ml of a 1 mg/ml dilution was filtered through a 5 ml syringe lined at the bottom with column packed with a cut piece of polypropylene (PP) disc. Next, the PP disc was washed with a dilute aqueous solution of sodium hydrogen carbonate (NaHCOa) to neutralize any excess HPA by conversion into the sodium salt. The material was allowed to air dry overnight before submitting for testing.

Antiviral Activity Testing

The concept of using chemically modified biopolymers was tested in different biological assays. Membrane disruption assays using haemolysis was used to demonstrate the membrane disruption activities of the modified polysaccharides and proof the concept in the laboratory. A genetically modified non-clinical HIV variant model, pseudo-HIV, was used to demonstrate the ability of membranedisrupting chitosan to inactivate an enveloped virus on a mask fabric coated with the cationic modified chitosan. Anti-SARS-CoV-2 activity was also demonstrated with a non-clinical variant, pseudo-SARS-CoV-2, on a commercially purchased mask coated with the cationic modified chitosan.

Proof of Concept Testing: Membrane disruption assays

The concept of using chemically modified biopolymers as membrane disruptors was investigated in a haemolytic assay where the greater the degree of haemolysis observed, the higher the membrane disruption potency of the test chemical. This is an acceptable assay to investigate the potential of the modified biopolymer to disrupt the membrane coat of an enveloped virus as the membrane is acquired as the virion emerges from an infected human cell.

Serial dilutions from 1 mg/ml to 0.125 mg/ml of HPA/CS, HTCC-CS and hydrophobically modified CS (HexD-CS) were prepared in PBS (pH 7.4). The samples were added to a suspension of 2% RBC in glass vials at a ratio of 4:1 and incubated at 37 °C for 4 h. Cells incubated in PBS pH 7.4, only, served as background sample. Triton X100 and PEI were used at 1% (v/v) each as positive controls. After incubation, the samples were centrifuged at 300xg at room temperature and the supernatants analysed for haemoglobin at an absorption wavelength of 570 nm on a Mettler Toledo UV5Bio spectrophotometer. Results were referenced to Triton X100, which was assumed to be 100%.

Demonstration of antiviral activity against enveloped viruses

Filtration of virus solution through modified chitosan bed

In general, dried materials of lauroyl- and stearoyl-chitosan were packed into 1 ml-syringes up to the 0.1 ml mark (Figure 1 ). The packed syringe column (10) contains an upper layer of hydrophobically modified-material (12), and a bottom layer of a glass wool plug (14) with a 0.45 urn filter paper (16) in between. The materials were sterilized with 70% ethanol before and after loading into the syringe column. The packed columns were then placed in an oven overnight at 30 °C to dry.

Antiviral activity of hydrophobically modified polysaccharides was initially tested by passing an aqueous buffer solution of pseudo-HIV particles (18) through a packed bed of the modified polysaccharides (12). The eluent (20) was then used to infect susceptible human cells (22) as previously described by Alexandre et al. (Alexandre, KB et al., 2010).

Filtration of virus solution through modified chitosan-coated fabrics

The modified chitosans were tested by passing an aqueous buffer solution of pseudo-HIV particles through a syringe lined at the bottom with cationic chitosan-coated mask fabric material. Uncoated mask fabric material was used as control. The filtrate was used to infect virus-susceptible human cells as previously described by Alexandre et al. 2010. We also compared the antiviral activity to that offered by the N95 respirator material, which are generally regarded as offering the best protection.

Demonstration of anti-SARS-CoV-2 of commercial mask coated with polycationic chitosan HTCC-CS was used in the real-life demonstration by coating onto commercially available masks. Non-medical masks were purchased from a local manufacturer and coated with HTCC-CS polycationic chitosan. Uncoated masks were used as controls. The specific testing protocol is described below:

A 96 well plate was used, and the experiment was done in triplicates. Cell control as well as virus control wells were included. To cell control wells, 150 pl of growth media was added and to the other wells, 100 pl of media was added. The masks were incubated with 400 pl of SARS-CoV-2 for an hour at room temperature. Afterwards, 50 pl of the flow through was added in triplicates to the 96 well plate. This was followed by the addition of 30 000 cells/100 pl/well of Vero cells and centrifugation at 3500 rpm for 3.5 hrs. After centrifuging, the experiment was incubated at 37 °C, 5% CC and 95% humidity for 72 hrs.

After 72 hours, 150 pl of media was removed from the plate and 100 pl of Bright-Glo luciferase substrate was added to all wells. This was followed by 2 min incubation at room temperature. After 2 min, 150 pl was transferred to a black plate and luminescence was read using TECAN Infinite F500 Luminometer.

Coating Methods

Further testing of the antiviral activity of a selection of the functionalized polysaccharide materials non-permanently incorporated into PPE fabrics is described thus. In a typical procedure, 100 mg of each chitosan-based material was dispersed into 10 ml of a suitable solvent (water and/or alcohol) and slowly stirred to attain a uniform solution.

The non-woven polypropylene (PP) was immersed in the modified polysaccharide formulation solution to ensure complete contact and wide penetration into the fabric fibers. The solution was shaken overnight and afterwards the fabric was removed from the solution and dried at room temperature overnight. Antiviral testing was conducted as previously described and depicted above. 3. Results

Table 1. Representative illustrations of typical chemical modification products of chitosan, cellulose and starch using long-chain fatty acyl, alkyl and quaternary

All the tested modified chitosan materials showed significant membrane disruption capability at 1 mg/ml, even higher than the polycationic positive control PEI (Figure 2). Even at half of this concentration, i.e., at 500 pg/ml, HPA/CS showed equivalent haemolysis to PEI.

The cationic chitosan salt HPA/CS (Method 08 synthetic product above) and the quaternary chitosan HTCC-CS (Method 07 synthetic product above) showed almost 80% membrane disruption of red blood cells compared to almost no membrane disruptive activity by unmodified chitosan at 1 .0 mg/ml (not shown in Figure 2). The hydrophobically modified chitosan HexD-CS (Method 04 synthetic product above) showed an almost identical membrane disruption activity compared to the cationic chitosan (HTCC-CS). This was taken as confirmation that the cationic and hydrophobically modified biopolymers were equivalently effective as membrane disruptors. All the materials tested by virus filtration assay showed greater than 60% retention of virus particles. Up to 95% virus particle removal was observed for the polypropylene impregnated with cationic chitosan.

The result of the antiviral assay of coated mask materials, using pseudo-HIV, is presented in Figure 3. While the N95 reference was only effective in inhibiting about 65-70% of the active viral particles from penetrating, the cationic chitosans demonstrated more than 90% efficacy when coated onto polypropylene and wool.

Demonstration of anti-SARS-CoV-2 of polypropylene mask fabric coated with polycationic chitosan.

The HPA/CS and HTCC-CS cationic chitosan antiviral activity were demonstrated with pseudo-SARS-CoV-2 after coating onto polypropylene using the same coating protocol described for the anti-HIV demonstration above. The result presented in Figure 4 shows that the quaternary chitosan HTCC-CS was superior and inhibited almost 100% of active viral particles from penetrating the coated polypropylene fabric.

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