Biomaterial with simultaneous antibacterial and antiviral properties

Abstract

The present invention relates to a composition comprising (poly)lysine and a mixture of λ-carrageenan and κ-carrageenan, wherein preferably the (poly)lysine is selected from the group consisting of: α-poly-L-lysine, ε-poly-L-lysine, α-poly-D-lysine, ε-poly-D-lysine, and mixtures thereof. The present invention also relates to the composition as defined above as a biocidal product, and also to the composition as defined above for use for the prevention of viral infections and / or bacterial infections and / or fungal infections.

Description

[0001] BIOMATERIAL WITH SIMULTANEOUS ANTIBACTERIAL AND ANTIVIRAL

[0002] PROPERTIES

[0003] The present invention concerns a biomaterial with simultaneous antibacterial and antiviral properties. The present invention also concerns uses of said biomaterial, in particular for antibacterial and antiviral applications.

[0004] Bacterial and viral infections are a major health problem worldwide and spreading of microbes mediated by contact with surfaces or skin is one of the leading cause of infection diffusion (Lax S, Gilbert JA. Hospital-associated microbiota and implications for nosocomial infections. Trends Mol Med. 2015 Jul;21 (7):427-32. doi: 10.1016 / j.molmed.2015.03.005. Epub 2015 Apr 20. PMID: 25907678). COVID-19 pandemic and the increasing rate of antibiotic-resistant bacteria point out the importance of reducing surface-mediated transmission using innovative technologies (Cohen MS, Corey L. Combination prevention for COVID- 19. Science. 2020 May 8;368(6491):551. doi: 10.1126 / science.abc5798. PMID: 32381692; van Doremalen N, Bushmaker T, Morris DH, Holbrook MG, Gamble A, Williamson BN, Tamin A, Harcourt JL, Thornburg NJ, Gerber SI, Lloyd-Smith JO, de Wit E, Munster VJ. Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1 . N Engl J Med. 2020 Apr 16;382(16): 1564-1567. doi: 10.1056 / NEJMc2004973. Epub 2020 Mar 17. PMID: 32182409; PMCID: PMC7121658; and Li B, Webster TJ. Bacteria antibiotic resistance: New challenges and opportunities for implant-associated orthopedic infections. J Orthop Res. 2018 Jan;36(1):22-32. doi: 10.1002 / jor.23656. Epub 2017 Aug 11. PMID: 28722231 ; PMCID: PMC5775060). Thus, new approaches have been recently developed to overcome microbe resistance and virulence factors spreading that often follow new antimicrobial drugs commercialization. Notably, new materials loaded with antibiotics to counteract bacterial infections have been proposed as an alternative way to locally deliver bactericidal molecules, thus reducing systemic side effects and antibiotic overall concentration (Musella M, Guido A, Musella S. Collagen tampons as aminoglycoside carriers to reduce postoperative infection rate in prosthetic repair of groin hernias. Eur J Surg. 2001 Feb;167(2):130-2. doi: 10.1080 / 110241501750070592. PMID: 11266253). However, this type of technology does not generally overcome antibiotic-resistance risks. In fact, other cutting-edge biomaterials have been developed to have an antimicrobial effect using metal coatings, such as gold, iron or silver, amongst others (Vasilev, K., Cavallaro, A., & Zilm, P. (2018). Special Issue: Antibacterial Materials and Coatings. Molecules (Basel, Switzerland), 23(3), 585. https: / / doi.org / 10.3390 / molecules23030585). Nonetheless, these effective antimicrobial agents might lead to an increased cytotoxicity at highest concentrations and the accumulation of antibacterial metals in human body still has to be fully investigated in terms of safety (AshaRani, P. V., Low Kah Mun, G., Hande, M. P., & Valiyaveettil, S. (2009). Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS nano, 3(2), 279-290. https: / / doi.org / 10.1021 / nn800596w). Thus, a new antimicrobial biomaterial is needed.

[0005] The aim of the present invention is to provide a new antimicrobial biomaterial having also an antiviral activity.

[0006] Another aim of the present invention is also to provide a composition in the form of a hydrogel having antimicrobial and antiviral activities, and also preferably an antifungal activity.

[0007] Another aim of the present invention is to provide antiviral and antibacterial coatings and hydrogels, preferably also having an antifungal activity.

[0008] Therefore, the present invention relates to a composition comprising (poly)lysine and a mixture of A-carrageenan (lambda-carrageenan) and K- carrageenan (kappa-carrageenan).

[0009] Carrageenans are red algae produced polysaccharides that can be found in 6 different forms (Chapter Ten - Pharmaceutical, Cosmeceutical, and Traditional Applications of Marine Carbohydrates; https: / / doi.org / 10.1016 / B978-0-12-800268- 1.00010-X). Three principal carrageenan forms are commercialized lambda- carrageenan, lota-carrageenan and kappa-carrageenan (A review of the nutrient composition of selected edible seaweeds; https: / / www.sciencedirect.com / topics / agricultural-and-biological-sciences / carrageenan). Kappa carrageenan has been used for its gel forming capabilities in food industry, whereas lambda carrageenan provides viscosity to colloidal systems without full gelling. As several polysaccharides possess antiviral properties, carrageenans have been also reported to have antiviral activity and to show different inhibitory effects on different viruses (The Antiviral Activities and Mechanisms of Marine Polysaccharides: An Overview, Mar. Drugs 2012, 10, 2795-2816; doi:10.3390 / md10122795).

[0010] Having individual molecules that can exhibit both antimicrobial and antiviral properties generally prove to be impossible. However, supramolecular assemblies of multiple biomaterials where the individual properties (antimicrobial, antiviral and antifungal) can be preserved, also in the form of a stable gel or coating, can achieve this goal.

[0011] As explained further below, the composition of the invention can be prepared in many various forms, such as a hydrogel. It can be used also as a coating formulation. This composition may be prepared and formulated for example as a film, a hydrogel, a bioink, a viscous gel or a viscous hydrogel.

[0012] The composition according to the invention is not toxic.

[0013] The composition according to the invention is also advantageous in that it has a surprising synergistic antimicrobial and / or antiviral activity, and also in that it has synergistic mechanical properties.

[0014] The composition according to the invention is also advantageous in that it can be prepared in the form of a stable hydrogel having surprisingly improved mechanical properties.

[0015] The composition according to the invention is also advantageous in that it has a satisfying minimum contact time to observe biocidal activity from 1 sec to 3 hours, preferably from 1 s to 30 minutes, as well as a satisfying maximal biocidal activity, preferably from 2log to 6log reduction.

[0016] The composition can be applied to both living tissues and non-living material (fomites).

[0017] The composition according to the invention thus comprises at least three components: (poly)lysine, A-carrageenan and K-carrageenan.

[0018] The composition according to the invention may also comprise water and is preferably an aqueous composition. According to this embodiment, the aqueous composition may comprise at least one salt, in particular it may comprise T ris or PBS.

[0019] According to the invention, the term “(poly)lysine” refers to lysine or to polylysine. As lysine, one may mention either L-lysine or D-lysine. As polylysine, one may mention either L-polylysine or D-polylysine.

[0020] According to an embodiment, the composition according to the invention comprises at least one polylysine. Polylysine refers to a polymer of the structural unit lysine. Polylysine refers to poly-L-, poly-D- or poly-LD-lysine.

[0021] Poly-L-lysine is for example a positively charged synthetic polymer (produced in the form of a salt with a counterion. The counter ion may be selected from, but is not limited to, hydrochloride, hydrobromide or trifluoracetate. Preferably, the polylysine has a molecular weight comprised from 1 000 Da to 2 MDa, preferably from 1 200 Da to 15 000 Da, more preferably from 1 250 Da to 6 000 Da.

[0022] According to the invention, the polylysine as mentioned above consists of n repetitive units (or n lysine residues), n being preferably comprised from 1 to 500, more preferably from 10 to 100.

[0023] According to an embodiment, the (poly)lysine according to the invention is selected from the group consisting of: a-poly-L-lysine, e-poly-L-lysine, a-poly-D-lysine, e-poly-D-lysine, and mixtures thereof. Preferably, the (poly)lysine according to the invention is selected from the group consisting of: a-poly-L-lysine, e-poly-L-lysine, and mixtures thereof. More preferably, the (poly)lysine according to the invention is e-poly- L-lysine (or PeL).

[0024] According to a preferred embodiment, the (poly)lysine has the following formula (1): n being an integer comprised from 1 to 500, in particular from 10 to 100, preferably from 10 to 30.

[0025] According to an embodiment, the composition according to the invention comprises from 0.01 mg / mL to 500 mg / mL, preferably from 0.1 mg / mL to 100 mg / mL of (poly)lysine.

[0026] According to an embodiment, the composition according to the invention comprises from 20% to 65%, preferably from 30% to 60%, and more preferably from 35 to 55% by weight of (poly)lysine in relation to the total weight of said composition.

[0027] According to an embodiment, in the composition according to the invention, the ratio between the weight of A-carrageenan and the weight of K-carrageenan is from 2 / 98 to 50 / 50, preferably from 5 / 95 to 50 / 50, even more preferably from 1 / 20 to 1 / 1. Preferably, this ratio is 1. According to an embodiment, the composition according to the invention comprises from 1% to 4%, preferably from 1 % to 2%, by weight of K-carrageenan in relation to the total volume of said composition.

[0028] According to an embodiment, the composition according to the invention comprises from 1% to 4%, preferably from 1 % to 2%, by weight of A-carrageenan in relation to the total volume of said composition.

[0029] According to an embodiment, the composition according to the invention comprises from 0.05% to 5%, preferably from 1% to 3%, more preferably from 1.5% to 2%, by weight of the mixture of A-carrageenan and K-carrageenan in relation to the total volume of said composition.

[0030] According to an embodiment, the A-carrageenan and the K-carrageenan of the composition as mentioned above has a molecular weight greater than 200 kDa.

[0031] According to an embodiment, the A-carrageenan of the composition as mentioned above has a molecular weight comprised from 500 000 g.rnol'1to 2 000 000 g.rnol'1, preferably from 700 000 g.rnol'1to 1 200 000 g.rnol'1.

[0032] According to an embodiment, the K-carrageenan of the composition as mentioned above has a molecular weight comprised from 300 000 g.rnol'1to 1 500 000 g.rnol'1, preferably from 500 000 g.rnol'1to 1 000 000 g.rnol'1.

[0033] According to a preferred embodiment, the mixture of A-carrageenan and K- carrageenan as defined above in the composition of the invention is in the form of a hydrogel.

[0034] Hydrogels have several unique characteristic properties, including their similarity to tissue extracellular matrix, support for cell proliferation and migration, controlled released of drugs or growth factors, minimal mechanical irritation to surrounding tissue, and nutrient diffusion, that support the viability and proliferation of cells.

[0035] According to an embodiment, the composition as defined above is in the form of a hydrogel.

[0036] According to an embodiment, 2% kappa carrageenan can form stable hydrogels, and by inclusion of lambda carrageenan into such formulation at a ratio above 0.01 %, a gelling antiviral composition can be achieved. Preferably, the composition according to the invention comprises a hydrogel made of a mixture of A-carrageenan and K-carrageenan as defined above, said hydrogel being incorporated or loaded with (poly)lysine as defined above.

[0037] According to an embodiment, when the composition according to the invention is in the form of a hydrogel, said hydrogel has a loading concentration of (poly)lysine from 0.1 mg / mL to 100 mg / mL, preferably from 1 mg / mL to 50 mg / mL.

[0038] According to an embodiment, the hydrogel according to the invention has a loading time of (poly)lysine from 1 min to 90 hours, preferably from 1 hour to 48 hours.

[0039] According to the invention, the term “loading time” refers to the time of incubation of a formed hydrogel in a (poly)lysine solution of given concentration for a specified duration in room temperature.

[0040] According to an embodiment, the hydrogel according to the invention has a loading rate of (poly)lysine comprised from 30% to 100%, preferably from 40% to 90%, and more preferably from 45% to 60%.

[0041] Preferably, the hydrogel according to the invention has a loading rate of (poly)lysine comprised from 20% to 100%, preferably from 25% to 80%, and more preferably from 30% to 60%.

[0042] According to the invention, the term “loading rate” is expressed in percentage %) and refers to the amount of (poly)lysine loaded in the hydrogel in comparison with the total (poly)lysine present in the loading solution regardless the initial concentration of (poly)lysine used for loading. The term “loading solution” refers to the initial solution comprising the (poly)lysine for the preparation of the hydrogel, the initial solution corresponding to the solution of (poly)lysine wherein the carrageenan hydrogel is immersed for 24h to 48h in order to load the hydrogel with (poly)lysine.

[0043] This loading rate may be measured by using a trace protein detection kit for BC Assay Method according to S.q.Rao et a / work (LWT - Food Science and Technology 146 (2021) 111422; (BC Assay Protein

[0044] Quantification Kit, Advion Interchim Scientific).

[0045] According to an embodiment, the hydrogel according to the invention has a cumulative amount of released (poly)lysine comprised from 0 mg / mL to 10 mg / mL, preferably from 1 mg / mL to 8 mg / mL, and more preferably from 4 mg / mL to 8 mg / mL over 7 days. According to the invention, the term “cumulative amount of released (poly)lysine” refers to the amount of (poly)lysine released from the hydrogel network to the surrounding medium per unit of volume of the formed hydrogel.

[0046] The present invention also relates to the use of the composition as defined above in a cosmetic composition that can be in direct contact with hair and skin. The present invention thus also relates to a cosmetic composition comprising the composition as defined above.

[0047] The present invention also relates to the use of the composition as defined above as a coating. The present invention thus also relates to a coating comprising the composition as defined above that can be applied to living tissues and non-living surfaces.

[0048] The composition according to the invention is also used as a cleaning solution for lenses. The present invention also relates to a cleaning solution for lenses comprising the composition as defined above.

[0049] It may also be used as means for sexual protection. The present invention also relates to means for sexual protection comprising, or being coated with, the composition as defined above.

[0050] The composition according to the invention is also used as disinfectant. The present invention also relates to a disinfectant comprising the composition as defined above.

[0051] The composition according to the invention is also used for preparing a food packaging. The present invention also relates to a food packaging comprising, or being coated with, the composition as defined above.

[0052] As explained below, the composition according to the invention may be used in a medical device, preferably a biocidal product. The present invention thus also relates to a biocidal product including the composition as defined above. The composition according to the invention may be used for humans and also for animals.

[0053] The composition according to the invention thus has many application fields, such as tissue engineering products, cell carriers, cosmetic skin care and hair care products, drug delivery systems, topical infection treatment solutions and woundcare products, and other personal hygiene products (hand and body disinfectants, including sexual protection products).

[0054] The present invention also relates to a device, preferably a medical device, comprising a coating comprising a composition as defined above. A “device” herein refers to an object comprising at least one surface. In one embodiment, the composition according to the invention covers at least a portion of the surface of said device. In one embodiment, the surface of the device of the present invention comprises, consists of, or at least partly consists of metal such as titanium, plastic such as silicone, ceramic or other materials such as wood. In one embodiment, the above-mentioned surface is a living tissue, such as human or animal skin, human or animal hair or similar structures thereof including open and infection wounds. In one embodiment, the above-mentioned surface is the surface of a cosmetic product be it a packaging item, or an applicator in the form of sponges, thin membranes, small or large brushes or equivalents (such as liquid lipstick applicators or mascara applicators). In one embodiment, the composition according to the invention is a viscous solution which can be applied as a surface treatment without necessarily creating a coating in the form of a cream or a gel.

[0055] The present invention thus also relates to the use of the composition for coating a surface, including the application of said composition onto said surface. For example, said surface is a living tissue, such as human or animal skin, human or animal hair or similar structures thereof including open and infected wounds. According to another embodiment, said surface is a solid surface.

[0056] By “medical device” is meant herein items such as catheters, stents, endotracheal tubes, hypotubes, suture material, wound dressings, filters such as those for embolic protection, surgical instruments and the like. Any device that is typically coated in the medical arts can be used in the present invention. It is further in the scope of the invention, wherein the term refers to any material, natural or artificial that is inserted into a mammal. The term “wound dressing” refers hereinafter to any pharmaceutically acceptable wound covering, such as: a) films, including those semipermeable or a semi-occlusive nature such as polyurethane copolymers, acrylamides, acrylates, paraffin, polysaccharides, cellophane and lanolin; b) hydrocolloids including carboxymethylcellulose, protein constituents of gelatin, pectin, and complex polysaccharides including acacia gum, guar gum and karaya, which may be utilized in the form of a flexible foam or, in the alternative, formulated in polyurethane or, in a further alternative, formulated as an adhesive mass such as polyisobutylene; c) impregnates including pine mesh gauze, paraffin and lanolin- coated gauze, polyethylene glycol-coated gauze, knitted viscose, rayon, and polyester; and d) cellulose-like polysaccharides such as alginates, including calcium alginate, which may be formulated as non-woven composites of fibers or spun into woven composites. The present invention also relates to the composition as defined above as a drug. The present invention also relates to a medicament comprising the composition as defined above. The present invention also relates to a pharmaceutical composition comprising at least one composition as defined above, and also at least one pharmaceutically acceptable excipient.

[0057] The present invention also relates to a composition as defined above for use for the prevention of viral infections.

[0058] The present invention also relates to a composition as defined above for use for the prevention of bacterial infections.

[0059] The present invention also relates to a composition as defined above for use for the prevention of fungal infections (including infections due to fungus or yeast).

[0060] The present invention also relates to a composition as defined above for use for the prevention of sexually transmitted infections.

[0061] The present invention also relates to the use of the composition as defined above as an antiviral agent. The present invention also relates to the use of the composition as defined above as an antibacterial agent. The present invention also relates to the use of the composition as defined above as an antifungal agent. The present invention also relates to the use of the composition as defined above as a biocide agent.

[0062] The present invention also relates to a method for preventing viral infections, comprising the administration of a pharmaceutically amount of the composition as defined above to a patient in need thereof.

[0063] The present invention also relates to a method for preventing bacterial infections, comprising the administration of a pharmaceutically amount of the composition as defined above to a patient in need thereof.

[0064] The present invention also relates to a method for preventing fungal infections, comprising the administration of a pharmaceutically amount of the composition as defined above to a patient in need thereof.

[0065] As mentioned above, the present invention also relates to the prevention of viral infections either through fomites (non-living vectors) or person-to-person contacts (air-borne or physical) through prevention of viral infectivity by creating biocompatible gel based barriers on non-living surfaces or living surfaces such as oral, skin or vaginal epithelium. The composition according to the invention may be used as a film, a sponge or a bio-ink. According to the invention, a film is a thin continuous structure (for example from 100 nm to 10 pm) covering a given substrate. According to the invention, a sponge is defined as a 3D structure where more than 80% of the volume is composed of open, closed or interconnected pores induced through processes such as salt leaching, freeze-drying, and critical point drying. According to the invention, a bioink is defined as a liquid system composed of one or more polymeric and other components (such as ceramic or metallic nano / microparticles) which can harbor living cells and can be formed into solid structures (in the form of gels preferably) via an additive manufacturing process such as extrusion 3D printing.

[0066] By “preventing”, it is meant avoiding the viral and / or bacterial and / or fungal infection to occur.

[0067] The “subject” refers to any subject and typically designates a patient. In any case, the subject is preferably a vertebrate, more preferably a mammal (such as a domesticated animal or companion animal), even more preferably a human being.

[0068] Examples of viral infections are infections caused by a virus selected from the group consisting of: Severe Acute Respiratory Syndrome coronavirus-2 (SARS-COV- 2), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Herpes Simplex virus (HSV), Human Immunodeficiency virus (HIV), and Human Papillomavirus (HPV). Other viral infections may include Feline Calicivirus, Human influenza H3N2, Human influenza H1N1 viruses, and Rotaviruses.

[0069] Bacterial infections are caused by a bacterium such as a Gram+ bacterium or a Gram- bacterium.

[0070] Fungal infections are caused by a fungus, such as Aspergillus niger or Candida albicans, or by a yeast.

[0071] In one embodiment, the composition of the invention has more than 70% growth inhibition of at least one bacterium, more particularly, more than 75%, more than 80%, typically, more than 82, 84, 86, 88, 90, 91 , 92, 93, 94, 95, 96, 97, 98% or 99.999% growth inhibition of at least one bacterium.

[0072] In one embodiment, the at least one bacterium is a Gram-positive bacterium or Gram-negative bacterium, preferably Gram-positive bacterium.

[0073] In one embodiment, the Gram-negative bacterium is a Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Escherichia coli, Klebsiella pneumoniae, Enterobacter species or Legionella bacterium, preferably Escherichia coli or Pseudomonas aeruginosa. The following Gram-negative bacteria may also be mentioned: Chlamydia trachomatis, Treponema pallidum or Neisseria gonorrhoeae.

[0074] In one embodiment, the Gram-positive bacterium is a Staphylococcus, Micrococcus or Enterococcus bacterium, or Listeria monocytogenes.

[0075] Bacteria of the “Enterococcus" genus are the cause of important clinical infections such as urinary tract infections, bacteremia, bacterial endocarditis, diverticulitis, and meningitis.

[0076] In one embodiment, the Enterococcus is a vancomycin-resistant Enterococcus, such as E. faecalis or E. faecium.

[0077] Bacteria of the “Staphylococcus” genus, such as, for example, S. aureus, S. epidermidis, S. capitis, S. caprae, S. haemolyticus, S. lugdunensis, S. schleiferi, S. simulans and S. warned are the main agents of infections on foreign materials for example in prosthetic joint infections.

[0078] Accordingly, in one embodiment the Staphylococcus is selected from S. aureus, S. epidermidis, S. capitis, S. caprae, S. haemolyticus, S. lugdunensis, S. schleiferi, S. simulans and S. warned, preferably S. aureus and S. epidermidis, more preferably S. aureus.

[0079] Bacteria of the “Micrococcus" genus are generally thought to be a saprotrophic or commensal organism, though it can be an opportunistic pathogen, particularly in hosts with compromised immune systems, such as HIV patients. Micrococci are normally present in skin microflora, and the genus is seldom linked to disease.

[0080] In one embodiment, the Micrococcus is a M. luteus bacterium.

[0081] The present invention also relates to a method for preparing the composition as defined above, in particular as a hydrogel, comprising the following steps:

[0082] - preparing the mixture of A-carrageenan and K-carrageenan, for example by mixing A-carrageenan and K-carrageenan powders in an aqueous solution (possibly comprising a buffer solution); optionally adding a salt to said mixture, in order to form a hydrogel, and

[0083] - adding a (poly)lysine solution to said mixture, in order to obtain the composition.

[0084] According to an embodiment, after the addition of the salt, the method comprises a step of formation of the gel at room temperature, for example for at least 15 minutes. According to an embodiment, the salt is selected from the group consisting of sodium, calcium or potassium sulfates or phosphates, or any physiological buffer solution such as PBS. Preferably, the salt is calcium chloride or potassium chloride.

[0085] According to an embodiment, the salt is a solution of CaCh or KCI.

[0086] Preferably, the first step for preparing the mixture includes a heating step, for example at a temperature from 70°C to 90°C, preferably around 80°C.

[0087] According to an embodiment, the mixture is prepared by mixing A-carrageenan and K-carrageenan powders in water or in a buffer solution, such as Tris / NaCI buffer or DPBS buffer.

[0088] Once the powders are dissolved, preferably, a salt solution, such as CaCh or KCI, is added into the above-mentioned mixture. Preferably, the mixture and the salt are left under stirring, for 10 minutes to 2 hours, and in particular at a temperature from 70°C to 90°C, preferably at 80°C.

[0089] The step of adding the (poly)lysine solution is carried out for example by preparing such solution by dissolving (poly)lysine solution into water or a buffer solution as mentioned above, and then by adding said solution onto the hydrogel as mentioned above, and then the hydrogel with the (poly)lysine is left for example for 24h to 48h at room temperature. The aforementioned (poly)lysine solution has an adjusted concentration to ensure optimal loading which corresponds to 5 mg / ml to 7.5 mg / ml.

[0090] The present invention also relates to a method for preparing the composition according to the invention in the form of a film. Such method comprises the preparation of solutions of lambda and kappa carrageenan solution on one side and the preparation of a (poly)lysine solution on the other side. Then, these solutions are applied either by an iterative process of subsequent dipping of the material or via concomitant spraying of both solutions.

[0091] The present invention also relates to a method for preparing the composition according to the invention in the form of a sponge. According to this embodiment, the hydrogel is prepared as described above, and then the method comprises freezing the formed gel at a temperature comprised from -20°C to -80°C, followed by lyophilisation up until compete removal of the aqueous component.

[0092] The present invention also relates to a method for preparing the composition according to the invention in the form of a bioink. According to this embodiment, the pre-formed gels are used under extrusion printing, gel viscosity can be adjusted for proper extrusion followed by crosslinking or coagulation. FIGURES

[0093] Figure 1 : Antiviral activity of negatively-charged polysaccharides in solution with SARS-CoV-2 virus-like particles on Vero cells.

[0094] Figure 2: Antiviral activity of negatively charged polysaccharides in solution against SARS-CoV-2 virus-like particles in contact with A) Vero cells and B) CALU-3 cells.

[0095] Figure 3: Evaluation of the antiviral activity of carrageenans lambda (-Car) of various MW (from 53 to 1 ,110 kDa) against virus-like particles and their infectivity on Vero cells.

[0096] Figure 4: Antiviral activity of diverse carrageenan hydrogels combinations against SARS-CoV-2 virus-like particles in contact with Vero cells.

[0097] Figure 5: Antiviral activity of Carrageenan-based hydrogels loaded with PeL polycations (n=3, triplicate).

[0098] Figure 6: Antibacterial and antifungal activities of Carrageenan based hydrogels loaded with PeL polycations (n=3, triplicate).

[0099] Figure 7: Long-term antibacterial activity of Carrageenan-based hydrogels loaded with PeL polycations against E.Coli and S. Aureus strains (n=3, triplicate).

[0100] Figure 8: Time-kill curves of a) E. Coli and b) S. aureus and inactivation curves of c) E. Coli and d) S. aureus exposed to Car hydrogels loaded with PeL polycations.

[0101] Figure 9: Biocompatibility assays of loaded Car hydrogels. A) Cell viability after 24h incubation with extracts of hydrogels and (B) Cell viability after 24h incubation in direct contact with hydrogels. The representative microscopy images of Balb 3T3 cells after 24h of incubation are also presented (n=3, triplicate).

[0102] Figure 10: Rheological behavior of carrageenan-based hydrogels loaded or not with PeL polycations as a function of frequency (0.01 to 10 Hz) at 25°C.

[0103] Figure 11 : Evaluation of cumulative amount of released polylysine (mg / mL) from Car hydrogels discs in buffer solutions using BC kit method. EXAMPLES

[0104] Materials

[0105] A-Carrageenan (L-Car) and k-Carrageenan (k-Car), calcium chloride anhydrous (CaCh), sodium chloride (NaCI) and potassium chloride (KCI) were purchased from Sigma-Aldrich (Saint-Louis, Missouri, USA). Chondroitin sulfate A (CSA) and fucoidan (FUC) were purchased from Sigma-Aldrich (Saint-Louis, Missouri, USA). Poly epsilon-L-Lysine HCI (PeL) (MW = between 3.5 and 4.5 KDa) was purchased from Biosynth Carbosynth (Staad, Switzerland). Tris (hydroxymethyl)-aminomethane (Tris), thiazolyl blue tetrazolium bromide (MTT), and Mueller Hinton (MH) broth medium were purchased from Merck KGaA (Darmstadt, Germany). Dulbecco’s modified Eagle medium (DMEM High Glucose), DMEM, high glucose with HEPES and 100X penicillin-streptomycin were purchased from Dominique Dutscher (Bernolsheim, France) and fetal bovine serum was purchased from Thermo Fischer Scientific (Waltham, Massachusetts, USA). Mouse Balb T3T cells (ATTC CCL163) and Vero cell lines (ATTC CCL-81) were obtained from ATCC (Manassas Virginia, USA).

[0106] NATURAL POLYSACCHARIDES SCREENING FOR ANTIVIRAL ACTIVITY

[0107] Several negatively-charged polysaccharides were reported for their antiviral activity: dextran sulfate (DEX5), hyaluronic acid (HA108), sodium alginate (ALG), chondroitin sulfate A (CSA), fucoidan (FUC), heparine (HEP), iota-carrageenan (i- Car), kappa-carrageenan (k-Car), lambda-carrageenan (L-Car) and chitosan.

[0108] The Antiviral activity and biocompatibility of these polysaccharides were evaluated to select polysaccharide with the best antiviral activity and no cytotoxicity.

[0109] Antiviral activity of negatively-charged polysaccharides in solution

[0110] A stock solution of dextran sulfate (DEX5), hyaluronic acid (HA108), sodium alginate (ALG), chondroitin sulfate A (CSA), fucoidan (FUC), heparin (HEP), iota- carrageenan (i-Car), kappa-carrageenan (k-Car), lambda-carrageenan (A-Car) and chitosan were prepared by dissolving the polysaccharides powder in the DMEM / FBS medium overnight. The stock solutions were then diluted from 200 pg / mL to 0 pg / mL into a suspension of SARS-CoV-2 virus-like particles (SARS-CoV-2 vIp). Viral suspension was kept in contact with different concentrations of negatively charged polysaccharides for 1 h at 37°C and, was then put in contact with Vero cells monolayer (monkey kidney epithelial cells) for 24h. To confirm the results obtained with SARS- CoV-2 vIp, replicated SARS-CoV-2 (P3) was kept in contact with different concentrations of negatively charged polysaccharides for 1h at 37°C. Then, the supernatant was put in contact with CALll-3 cells (Human Lung Adenocarcinoma Cell line) for24h. To assess the viral infection rate on cells, the presence of the luciferases enzymes was quantified.

[0111] These negatively charged polysaccharides, most of them originated from sea resources, have been tested for their potential antiviral activity against SARS-CoV-2. Each polysaccharide, tested from 0 pg / mL to 200 pg / mL, have been incubated with a suspension of SARS-CoV-2 virus-like particles (SARS-CoV-2 vIp). The infection rate is then calculated and compared to negative control (untreated viruses = 0 pg / mL). The results show that hyaluronic acid (HA108), sodium alginate (ALG), chondroitin sulfate A (CSA), fucoidan (FUC), and Heparine (Hep) did not show any strong inhibitory activity against SARS-CoV-2 vIp (Figure 1). Although, it has been described that some of them presented antiviral properties against viruses (Andrew, M., & Jayaraman, G. (2021). Marine sulfated polysaccharides as potential antiviral drug candidates to treat Corona Virus disease (COVID-19). Carbohydrate research, 505, 108326. Mehwish Jabeen, Melody

[0112] Dutot, Roxane Fagon, Bernard Verrier, Claire Monge. Seaweed Sulfated Polysaccharides against Respiratory Viral Infections. Pharmaceutics, MDPI, 2021 , 13 (5), pp.733. 10.3390 / pharmaceuticsl 3050733. hal-03434587; Jabeen, M., Dutot, M., Fagon, R., Verrier, B., & Monge, C. (2021). Seaweed Sulfated Polysaccharides against Respiratory Viral Infections. Pharmaceutics, 13(5), 733. https: / / doi.org / 10.3390 / pharmaceutics13050733). However, carrageenan polysaccharides presented a strong antiviral activity, even at low concentrations (Figure 1). The three different types of commercialized carrageenans (A review of the nutrient composition of selected edible seaweeds, were tested: kappa carrageenan (k-Car), iota carrageenan (i-Car) and lambda carrageenan (L-Car). The results show that L-Car exhibited the strongest antiviral activity against SARS-CoV-2 vIp, with infection decrease of more than 80% at a concentration of 100 pg / mL. Moreover, k-Car and i-Car were, to lesser extent, also active against SARS-CoV-2 vIp. The main difference between these three types of carrageenans is sulfate groups number. In fact, L-Car has the most sulfate groups per monomer (3 groups for L-Car, 2 groups for i-Car and 1 group for k-Car), which confirm the correlation between the antiviral activity and the degree of sulfation of carrageenan polysaccharide (Alvarez- Vihas, M., Souto, S., Florez-Fernandez, N., Torres, M. D., Bandin, I., & Dominguez, H. (2021). Antiviral Activity of Carrageenans and Processing Implications. Marine drugs, 19(8), 437. https: / / doi.org / 10.3390 / md19080437).

[0113] For dextran sulfate, despite that this polysaccharide showed promising antiviral activity, it has no ability to form hydrogels. In addition, dextran sulfate is a chemically modified natural polysaccharide. Therefore, the use of carrageenans, a natural polysaccharide, is more promising.

[0114] In order to confirm the antiviral activity of L-Car on SARS-CoV-2 vIp, chitosan (Chi50), a polysaccharide known to present antiviral properties was tested (Mar. Drugs 2012, 10, 2795-2816; doi:10.3390 / md10122795; J Appl Microbiol. 2021 ;00:1- 18 DOI: 10.1111 / jam.15202). The antiviral activity of chitosan was evaluated on SARS-CoV-2 vIp in Vero cells as a control (figure 2A). As expected, chitosan was not able to inhibit SARS-CoV-2 infection in Vero cells. This is probably due to the cationic nature of chitosan (J Appl Microbiol. 2021 ;00:1-18. DOI: 10.1111 / jam.15202), which prevent its binding with the positively charged spike protein (J Appl Microbiol. 2021 ;00:1-18. DOI: 10.1111 / jam.15202) and thus it cannot prevent interaction with host cells.

[0115] In addition, more antiviral tests were performed using different cell line: the CALll-3 cells, a commonly used respiratory cells line. Viral infection by SARS-CoV-2 vIp were tested on CALll-3 cells in the presence of L-Car and dextran sulfate (DEX5) (figure 2B). Results show that as well as for Vero cells, inhibition of viral infection on CALll-3 was observed at low concentrations of L-Car (IC50 = 71.89 pg.mL'1).

[0116] Antiviral activity of L-Carrageenan of different molecular weights

[0117] Full-length L-Car molecular weight is 1 ,110 kDa. To elaborate L-Car with different length of chains, these full-length L-Car chains were hydrolyzed at 60°C in HCI solution (0.1 M) for limited period of time. Finally, L-Car of different molecular weights were obtained: 52, 80, 159, 297, 321 , 360, 453 and 566 kDa with high monodispersity. Antiviral activity of L-Car of different molecular weights on Vero cells was evaluated as described above. The molecular weight of polysaccharides is a key parameter to consider for antiviral activity assessment (Mar. Drugs 2012, 10, 2795-2816; doi:10.3390 / md10122795; Recent advances in antiviral activities and potential mechanisms of sulfated polysaccharides, ). Therefore, the effect of L-Car molecular weight on the antiviral activity was investigated. The infection of Vero cells in contact with SARS-CoV-2 virus-like particles was evaluated in the presence of various concentrations of L-Car of different chain length (figure 3). Results show that L-Car with high molecular weights presented the best antiviral activity: 453 kDa L-Car, 566 kDa L-Car and full-length L-Car of 1.110 kDa. Therefore, full-length L-Car seems to be the most promising compound. However, L-Car have no gelling ability (no gelling polymer) while k-Car and i-Car being able to form gel. To elaborate hydrogels, a mixture of L-Car with k-Car or i-Car was used.

[0118] Antiviral activity of L-Carrageenan polysaccharide against viruses inducing STIs

[0119] Inhibition assays of VIH-1 infection

[0120] P4-R5 MAGI cells (HeLa cell line expressing CD4+ and CCR5+ receptors) were cultured 24h prior to infection at a density of 1 *104cells / well in a 96-well plate. A stock solution of L-Carrageenan was prepared by dissolving the polysaccharides powder in DMEM medium. The stock solution was then diluted (5, 1 , 0.5, 0.2,0.15; 0.1 ; 0.05 and 0.025 mg / mL) in DMEM medium and 10OpI of dilutions was added per well as antiviral agent (L-Carrageenan). HIV-1 virus suspension (100 mL / well) was added in the absence or presence of antiviral agent. After 24h of incubation at 37°C, 5%CC>2, the supernatant was removed and cells were washed twice with PBS buffer. To assess the HIV-1 infection on cells, the presence of the B-galactosidase enzymes was quantified by adding 200pl of buffer (Tris 1M pH8, 4-methylumbelliferyl-p-d- galactoside, p-Mercaptoethanol, Triton10%, H2O) and incubated for an additional 24h at 37°C, 5%CO2.

[0121] The fluorescence was evaluated at wavelengths of 360nm and 460nm using fluorescence Multi-Well Plate Reader (CytoFluor® 4000 series, UK). For each condition, relative fluorescence units (RFU) were measured and normalized to establish the inhibition of virus infection percentage (%). Results of antiviral activity were expressed by the half maximal effective concentration (EC50) calculated using SigmaPlot® 11.2 software. Inhibition assays of HSV-2 infection

[0122] The antiviral activity was evaluated by plaque reduction assay. Vero cells were seeded in 24-well plate and grown to 80% confluency for 24h. Different 10-fold dilutions of HSV-2 virus in DM EM were prepared from the stock viral suspension (from 10’1to 10-7).

[0123] The monolayers of Vero cells were infected with 200pL of different dilutions of HSV-2 viral suspension (10-4, 10’5, 10’6, 10'7) and incubated at 37°C, 5% CO2 for 1h to allow the virus to attach and entry into cells. Mixture solutions of different concentration of L-carrageenan prepared from stock solution diluted in DMEM (5, 1, 0.5, 0.2,0.15; 0.1 ; 0.05 and 0.025 mg / mL) with agar media (0.3% agarose / DMEM: 1 :1 (V / V)) were prepared. The viral inoculum was next removed from Viro cells monolayer and each well was directly overlaid with 1mL of the mixture solutions. Vero cells were then incubated at 37°C, 5% CO2 until lysis plaques appear (incubation at least for 72h). Acyclovir was used as positive control (EC50 (HSV-2) = 0.3 pg).

[0124] After 72h of incubation, agar media was aspirated and cells were fixed with 1 mL of 4% formaldehyde for 15 min at room temperature (RT). After removal of the formaldehyde, the cells were stained with 0.5 mL of crystal violet for 15 min at RT. The plates were washed with water and dried at RT. HSV-2 titer was calculated through plaque counting and expressed as plaque forming unit (PFU). Infection percentages (%) were calculated and the ECsowas calculated using SigmaPlot® 11.2 software.

[0125] Results of the antiviral activity of L-Carrageenan polysaccharide are presented in table 1.1. L-carrageenan was active against human viruses HSV-2 and VIH- 1 tested on HeLa cells and Vero cells, respectively. L-Carrageenan concentration needed to inhibit 50% of the cytopathic effect was 10 pg / mL for HSV-2 and 10 ng / mL for HIV-1.

[0126] Results indicate L-Car polysaccharide inhibit the infection with human viruses HSV-2 and VIH-1 inducing STIs besides SARS-CoV-2 infection.

[0127] Table 1.1 : Antiviral activity of L-Carrageenan against SARS-CoV-2, HSV-2 and VIH-1 viruses DIVERSE CARRAGEENAN HYDROGEL COMBINATIONS

[0128] To further assess the best formulation for carrageenan-based hydrogels, hydrogels based on different combinations of three types of carrageenan: i-Car, k-Car and L-Car were prepared. In order to select the best carrageenan combination, mechanical properties and antiviral activity of hydrogels were evaluated.

[0129] Different combinations of the three types of carrageenan were prepared and evaluated: i-Car, k-Car and A-Car. In fact, carrageenans have the ability to form hydrogels. In solution, the k-Car and i-Car types self-associate into helical structures and form rigid or flexible gels respectively. The lambda type does not form helices and is a non-gelling polysaccharide, so it does not produce hydrogels, but it bears viscous properties (Pettinelli, N., Rodriguez-Llamazares, S., Bouza, R., Barral, L., Feijoo-Bandin, S., & Lago, F. (2020). Carrageenan-based physically crosslinked injectable hydrogel for wound healing and tissue repairing applications. International journal of pharmaceutics, 589, 119828. Lester C. Geonzon, Xinye Zhuang, Amos M. Santoya, Rommel G. Bacabac, Jingli Xie, Shingo Matsukawa, Gelation mechanism and network structure of mixed kappa carrageenan / lambda carrageenan gels studied by macroscopic and microscopic observation methods, Food Hydrocolloids, olume 105, 2020, 105759, ISSN 0268-005X, https: / / doi.Org / 10.1016 / j.foodhyd.2020.105759).

[0130] Several hydrogel formulations have been synthetized and tested. Hydrogels composed of k-Car are more rigid and keep their structure when cut in different diameter discs. Hydrogels composed of i-Car are less rigid and very fragile. Besides, during the formulation, two salts were added to promote gelation of the carrageenan- based hydrogel, KCI or CaCh, as indicated in the literature (Viet T.N.T. Bui, Bach T. Nguyen, Taco Nicolai, Frederic Renou, Mobility of carrageenan chains in iota- and kappa carrageenan gels, Colloids and Surfaces A: Physicochemical and Engineering Aspects, Volume 562,2019, Pages 113-118, ISSN 0927-

[0131] 7757, https: / / doi.Org / 10.1016 / j.colsurfa.2018.11.017; Cynthia Fontes-Candia, Anna Strom, Patricia Lopez-Sanchez, Amparo Lopez-Rubio, Marta Martinez-Sanz, Rheological and structural characterization of carrageenan emulsion gels, Algal Research, Volume 47, 2020, 101873, ISSN 2211-9264, https: / / doi.Org / 10.1016 / j.algal.2020.101873). Thus, as k-Car hydrogels were found to be the most easy-to-handle in terms of rigidity and stability, all the formulations must contain at least 1 % (w / v) of k-Car. Depending on the application, the stiffness of the hydrogel and the concentration of k-Car can advantageously be modified to obtain a wide range of hydrogels with different rigidities. In order to select the best carrageenan combination, mechanical properties and antiviral activity of carrageenan hydrogels were evaluated.

[0132] Table 1. Mechanical properties of diverse carrageenan hydrogels combinations

[0133] Table 1 gives some parameters for the mechanical properties of hydrogels, such as the storage modulus (G’) and the loss modulus (G”). This table shows that the values of G’ and G” decrease for the mixture of k-Car and L-Car (in comparison for example with k-Car alone), but it is shown later (see Figure 10) that the combination of the carrageenans with the polylysine surprisingly gives increased values for G’ and G”.

[0134] Moreover, several compounds and mixtures were tested: kappa-carrageenan alone (k-Car 2%), as well as a mixture of kappa-carrageenan and iota-carrageenan (k-Car 1% / i-Car 1 % (k-Car / i-Car 2%)) and a mixture of kappa-carrageenan and lambda-carrageenan (k-Car 1 % / L-Car 1% (k-Car / L-Car 2%)) were tested for their antiviral properties. These three types of carrageenan-based hydrogels were exposed to SARS-CoV-2 vIp as previously described and the corresponding infection was evaluated on Vero cells. Our results show that the association between k-Car and L- Car at 2% (w / v) provided the best antiviral activity (figure 4).

[0135] Several MTT tests were performed on both Balb T3T and Vero cells to evaluate k-Car and L-Car at 2% (w / v) formulation. Results show no cytotoxicity on both cell lines, suggesting that k-Car / A-Car at 2% (w / v) is an interesting hydrogel base formulation to further assess antibacterial properties, once loaded with polycations.

[0136] PREPARATION OF CARRAGEENAN-BASED HYDROGELS

[0137] Carrageenan-based hydrogels were prepared by dissolving carrageenan powder (Car) at 80°C. A volume of 15.865 mL of distilled water or of a buffer solution (Tris / NaCI buffer (10mM / 150mM) or DPBS buffer (137.9 Mm NaCI), pH=7.4) was poured into a plastic heat-resistant lab jars (with lid) and put over a heating plate at 80°C. The carrageenan powders were weighted (167mg of L-Car and 167mg k-Car) and mixed in a 1.5 mL Eppendorf tube. Then, the carrageenan powder was gradually added in the heated water or buffer solution while stirring with a magnetic stirrer. After complete dissolution of the Car powder, 0.835 mL CaCh or KCI solution (5% (v / v), 50 mM) was added and stirred for 15 min. These carrageenan solutions were then poured into a pre-warmed petri-dishes (100 mm) and heated at 80°C in the oven (Memmert, germany) for 15 min. Finally, the petri dishes were left at room temperature (RT) for 15 min to obtain the carrageenan-based hydrogels. These hydrogels are made with a mixture k-Car / A-Car as carrageenan.

[0138] For further experiments, hydrogels were cut into 6 mm or 10 mm diameter discs and put in 48 or 24 well plate, respectively.

[0139] Four types of carrageenan-based hydrogels were prepared and are listed in table 2 according to the buffer solution and the salt used in hydrogel formulation. Based on visual observations, the gelling times were fast for all hydrogels (less than 1min). In order to measure the true gelling times of hydrogels, rheological measurements are needed to evaluate changes in viscoelastic properties of hydrogels over time. However, the gelling times of Car hydrogels were too fast and rheological measurement cannot be performed. Transparent or slightly white hydrogels were obtained after gelation at RT.

[0140] Carrageenan-based hydrogel loading with PeL

[0141] To provide antimicrobial properties to the Carrageenan-based hydrogels, they were cut and loaded with polylysine solution at different concentrations of Poly-e- Lysine (PeL). The PeL solutions were prepared by dissolving PeL powder in the same buffer solution used to prepare the Car hydrogels (distilled H2O, Tris / NaCI or DPBS buffer). A fixed volume of the PeL solution were then added to the Car discs. For antibacterial and antiviral assays, hydrogels were cut into 6 mm discs and put into 48 well plate. A volume of 225 pL of PeL solution is added to each well. For biocompatibility tests, 1.256 mL of PeL solution is added to 10 mm hydrogel discs in a 24 well plate. The well plates were sealed with plastic film of PeL solution and were covered with aluminum. T o provide a homogenous loading of Car hydrogels with PeL, well plates were placed on a shaker and left for 24h or 48h at RT. For each condition, three hydrogels were tested to perform technical replicates. Finally, Car hydrogels were washed at RT with the buffer solution (500 pL or 1.256 mL of buffer to wash hydrogels of 6mm or 10mm of diameter, respectively), 3 times for 5 min under stirring (shaker). Carrageenan hydrogel discs were loaded with different concentration of PeL polycations to provide antimicrobial activity. Antiviral activity, antibacterial properties and cytotoxicity of eighty-seven (87) formulations were evaluated; and eight formulations of carrageenan-based hydrogels loaded with PeL were selected (table 2). These hydrogels are made with a mixture k-Car / A-Car as carrageenan.

[0142] Table 2. Selected formulations of carrageenan-based hydrogels loaded with PeL.

[0143] Further characterization of carrageenan-based hydrogels

[0144] Composition of hydrogels

[0145] Table 3 shows the detailed description of hydrogels’ composition where results are expressed in percentage of the ingredient mass to hydrogel mass (%, W / W). For the PEL % (W / W) in the hydrogels given in table 3, it was estimated that all the amount of PEL in the initial solutions used for loading (5 or 7.5 mg / mL) was loaded in carrageenan hydrogels regardless the loading time (24h or 48h).

[0146] In general, the composition of hydrogels comprises 34% to 50% of PeL polycation, 15% to 21% of L-carrageenan, 15% to 21 % of K-Carrageenan and 6% to 15% of salt (KCI, NaCI or CaCh) depending on the hydrogel formulation. In some hydrogel formulations, the composition also comprises about 3% of Tris base. Table 3: Composition of dry carrageenan hydrogels (mass percentage % (w / w))

[0147] ANTIVIRAL ASSAYS

[0148] Antiviral assays were performed on carrageenan-based hydrogels loaded with polycations to evaluate their antiviral properties.

[0149] Antiviral activity of carrageenan-based hydrogels loaded with polycations

[0150] The antiviral activity of the eight selected formulations of carrageenan-based hydrogels were tested. Hydrogels of 6 mm diameter were put into a 48 well plate and 110 pL of the test inoculum containing SARS-CoV-2 virus-like particles were added per well / hydrogel. For each condition, three hydrogels were tested in order to have technical replicates. The inoculum was incubated at 37°C for 1 h while shaking. Then, 100 pL of the supernatant was added to 48 well plate containing monolayer of Vero cells at 104cells / well. After 24h of incubation at 37°C (under humidified atmosphere with 5% of CO2), the supernatant was removed and cells were washed once with PBS buffer. Vero cells were lysed using Gio Lysis Buffer, 1X (Promega, Madison, Wisconsin, USA) at 50pL / well. The lysis buffer was kept at RT for 10-15 min under stirring and 25 pl of lysate were transferred into a white plate. 50pL of Bright gio luciferase assay (Promega, Madison, Wisconsin, USA) was added to each well and fluorescence was evaluated after 10 sec with a Luminescence Plate Readers (Berthold Thoiry, France). For each condition, relative fluorescence units (RFU) were measured and normalized to establish the percentage of virus infection inhibition. Results are shown in figure 5 and the infection rate of Vero cells is compared to untreated viruses (infection control). The eight Car hydrogel formulations presented strong inhibitory activity against SARS-CoV-2 vIp. The antiviral activity of these hydrogels was ranged from 68 to 99.9999% of infection inhibition regardless the composition of car hydrogels and PeL concentration. These results show that the antiviral properties of carrageenan-based hydrogels were maintained after loading with PeL.

[0151] Antiviral activity of carrageenan based hydrogels loaded with PeL according to modified ISO 21702

[0152] The antiviral activity of Car hydrogels n4, n6, n7 and n8 have been tested under conditions defined by ISO 21702 (2019) adapted protocol for a contact time of 4 hours against Feline Calicivirus, Human influenza H3N2 and Human influenza H1N1 (table 4).

[0153] Hydrogels of 12 mm diameter were put into a 12-well plate and 440 pL of the test inoculum containing viruses were added per well / hydrogel. Positive control was a stainless-steel support. Replicate (3) samples of each hydrogel formulations were tested. The inoculum was incubated at 37°C for 4 hours under agitation. Then, 400 pL of the supernatant was added to 12 well plate containing monolayer of permissive cells (table 4) and incubated at 37°C under agitation.

[0154] Table 4: Cells and viral strains

[0155] After 90 minutes of incubation, the supernatant was removed and cells were washed three times with PBS buffer. Then, 550pL of Minimum Essential Medium (MEM) was added and incubated at 37°C, 5% CO2. When viral cytopathic effect appears in the infection control (CPE++) (15h post infection for Feline Calicivirus and 24h post infection for Human influenza H3N2 and H1N1), the supernatant was recovered in 10 mL of MEM for neutralization. Further dilutions 10’1, 10’2, 10’3, 10’4, 10-5, 10-6, 10-7and 10'8) of the neutralized mixture were prepared using MEM and added to a monolayer of permissive cells in 96-well plate.

[0156] The results have been determined by a visual reading of the cytopathogenic effects (CPE) and quantified by the TCIDso / ml technique on KER or MDCK+ cells according to Spearman-Karber method (Virology Methods Manual. 2007 Sep 2:25- 46. doi: The TCDIso is the dilution of a virus required to infect 50% of a given batch of inoculated cell cultures. Results of antiviral effect were converted to reduction log™ according to IS021702-2019 standard.

[0157] Table 5 presents the results of reduction Log™ against Feline Calicivirus, H3N3 and H1N1 according to adapted protocol ISO 21702- 2019.

[0158] Carrageenan-based hydrogels n4, n6, n7 and n8 were tested and results show that hydrogels n4, n6 and n8 were more effective against Feline Calicivirus and H1N1 viruses with a reduction log™ > 3 and a reduction log™ > 2, respectively. Hydrogels were less effective against H3N3 virus with a reduction log™ ^1 , where the antiviral activity was 49.88%, 36.90%, 68.38% and 90.00 % for hydrogels n4, n6, n7 and n8, respectively.

[0159] Table 5: Antiviral activity according to adapted protocol ISO 21702-2019 (reduction

[0160] Log™)

[0161] ANTIBACTERIAL ASSAYS AND ANTIFUNGAL ASSAYS

[0162] Carrageenan polysaccharides provided to hydrogels the antiviral activity. To provide them with antimicrobial properties, Car hydrogels were loaded with polylysine polypeptides. The antibacterial activity of Car hydrogels loaded with PeL polypeptides were evaluated to determine inhibition of bacterial growth in the supernatant. Staphylococcus aureus (S. aureus, ATCC 25923), Escherichia coli (E.coli, ATCC25922) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) strains were used to assess the antibacterial properties of the carrageenan-based hydrogels loaded with polycations. Bacterial strain was cultured aerobically at 37°C in a Mueller Hinton Broth (MHB) medium (Merck, Germany), pH 7.4. One colony was transferred to 10 mL of MHB medium and incubated at 37°C overnight, to provide a final density of 106CFU mL-1. To obtain bacteria in the mid logarithmic phase of growth, the absorbance at 620 nm (A620) of overnight culture was adjusted to 0.001 , corresponding to a final density of 8 x 105CFU mL"1.

[0163] Candida albicans (C. albicans, ATCC 18804) strain were used to assess the antifungal properties of the test samples. Bacterial strain was cultured aerobically at 37°C in a sabouraud-dextrose broth (SDB) medium and antifungal activity were evaluated according to the same protocol described above. As positive control, chlorohexidine (9 pL) was used. After 24h, the supernatant was removed and its absorbance at 620 nm was measured with a spectrophotometer.

[0164] Carrageenan hydrogels were cut in 6mm diameter discs and put into 48-well plate. They were loaded with different concentrations of PeL for a given time (24 to 48 hours) and sterilized with UV-light during 30 minutes. For each condition, three hydrogels were tested in order to have technical replicates. Then, 300 pl of bacterial culture (Ae2o = 0.001) were deposited in each well containing hydrogels, and incubated during 24 hours at 37°C. For negative control, empty wells were incubated at 37°C with bacterial culture only. For positive control, Tetracycline (10 pg / mL) and Cefotaxime (0.1 pg / mL) were added in S. aureus and E. coli culture, and chlorohexidine (9 pL) in P. aeruginosa culture. After 24h, the supernatant was removed and its absorbance at 620 nm was measured with a microplate reader (Allsheng AMR 100, China).

[0165] Results in figure 6 show that Car hydrogels loaded with PeL polycations provided excellent antimicrobial properties against bacteria Gram positive, S. Aureus, and bacteria Gram negative, E.Coli and P.Aeruginosa, with more than 99% of growth inhibition.

[0166] Antifungal properties of hydrogels were also evaluated against C. Albicans and results also show a growth inhibition of 99%. PeL polycations incorporated in Car hydrogels can provide good antibacterial and antifungal properties to these hydrogels. Poly-epsilon-lysin is known as a natural antimicrobial cationic peptide, which is FDA approved as safe (GRAS) as a food preservative (The Antimicrobial Mechanism of Action of Epsilon-Poly-L-Lysine However, the exact mechanism of PeL polypeptides as antibacterial agent against pathogens still not confirmed. It is reported that the antibacterial mechanism of PeL against E. coli may be attributed to disturbance on membrane integrity (Biochemical and Biophysical Research Communications 439 (2013) 148-153 http: / / dx.doi.org / 10.1016 / j.bbrc.2013.08.001). Using membrane models, M. Hyldgaard et al hypothesized that PeL destabilizes E.Coli membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles / micelles (The Antimicrobial Mechanism of Action of Epsilon- Poly-L-Lysine https : / / d oi . orq / 10.1128 / AEM .02204- 14) . It is also reported that the minimum fungicidal concentration (MFC) of PeL on Candida Albicans were 250 pg mL-1; and that PeL polypeptides inhibit the respiration and fermentation system of fungi and affect the plasma membrane disruption (Study of the mechanism of e-poly-l-lysine as an antifungal on Candida albicans and Saccharomyces cerevisiae https: / / doi. orq / 10.1016 / j.bbaqen.2022.130197).

[0167] Long-term antibacterial activity assays

[0168] S. aureus (ATCC 25923) and E. coli (ATCC25922) strains were used to assess the long-term antimicrobial effects of Car based hydrogel loaded with PeL according to previous work (Adv. Healthcare Mater. 2015, 4, 2026-2036; DOI: 10.1002 / adhm.201500546). Repetitive bacterial culture was performed in the presence of hydrogels for 7 days to assess their long-term antimicrobial effects. A volume of 300 pL of a midlogarithmic phase culture of bacteria was added (Ae2o = 0.001). Tetracycline (10 pg / mL) and Cefotaxime (0.1 pg / mL) were used as positive controls. The hydrogels were brought in contact with a fresh pathogen suspension for 24h at 37 °C. Every 24h, the supernatant was removed and replaced by a fresh suspension. Antibacterial activity was assessed by measuring absorbance at 620 nm with a spectrophotometer.

[0169] Results are shown in figure 7. Car hydrogels prepared with formulation 6 and 7 showed the best results against E.Coli strains with antibacterial effect lasting for 5 days and 6 days, respectively. Formulation 4, 5 and 8 preserved their antibacterial effect for almost 3 days. Antibacterial results against S. Aureus show similar results for formulations 4 and 5, an antibacterial effect for 3 days. Car hydrogels prepared according to formulations 7 and 8 presented a long-lasting of antibacterial effect of 6 days.

[0170] In terms of long-lasting antibacterial activity, hydrogels prepared according to formulation 6, 7 and 8 were the most effective against bacteria Gram positive and Gram negative.

[0171] Time-kill curve experiments

[0172] The time-to-kill curves were performed to assess the exposure time required to kill a standardized S.aureus (ATCC 25923) and E.coli (ATCC25922) inoculum (Pharmaceutics 2022, 14, 957 (disc); J. Med. Chem. 2021 , 64, 16480-16496, International Journal of Pharmaceutics 548 (2018) 431-442 (film)). Bacterial strains were prepared as described before. In 48 well plate, 300pL of bacterial cultures (A62o=O.OO1 corresponding to approximatively 8 * 105CFU mL"1) was added to Car hydrogels discs of 6mm of diameter, loaded with PeL. For each condition, three hydrogels were tested in order to have technical replicates. For negative control, empty wells were incubated at 37°C with bacterial culture only. After incubation at 37°C during 24 hours, aliquots of 100 pL of negative control and samples were taken from each well at Oh, 1 h, 3h and 6h and diluted in series in Mueller Hinton Broth (MH). Aliquots were then spread on two Mueller Hinton Agar (MHA) plates. The plates were incubated at 37°C for 24h and the viable colonies were evaluated. The kill curves were constructed by plotting the log™ CFU / rnl surviving at each time point in the presence and absence of Car hydrogels loaded with PeL.

[0173] Figure 8 shows the results of antibacterial activity expressed in log (CFU / rnL). The antibacterial activity of Car hydrogels loaded with polycations were evaluated against E.Coli (figures 8a and 8c) and S. Aureus (figures 8b and 8d) in time-kill curves up to 6h (these hydrogels are made with a mixture k-Car / A-Car as carrageenan). Bacteria were exposed to hydrogels loaded with PeL to determine the exposure time required to kill bacteria. The bacterial density at Oh was in the range of 4.9 to 6.3 x105CFU / mL. As expected, no decrease in the log (CFU / mL) was observed for the negative control (bacteria with no treatment). Escherichia coli is a Gram-negative bacterium, which might be responsible of urinary tract infections. Car hydrogels loaded with PeL had fast bactericidal effect on E.Coli. Hydrogels presented 3 bacteria log reduction after 1h of incubation (99.9% reduction) for almost all formulations. In addition, in all formulations of Car hydrogels, there was decreased log (CFU / mL) equal to zero after only 3h of incubation and bacteria log reduction increased to 6.

[0174] Staphylococcus aureus is a Gram-positive bacterium part of human skin flora and it might be responsible for severe diseases and multidrug resistant (MDR) infections. Car hydrogels loaded with PeL presented 2 bacteria log reduction after 3h of incubation (99% reduction) for almost all formulations. After 6h of incubation, bacteria log reduction increased, mostly up to 3.

[0175] DIRECT AND INDIRECT BIOCOMPATIBILITY ASSAYS

[0176] Biocompatibility of carrageenan-based hydrogels loaded with polycations was also evaluated. Direct and indirect in vitro cytotoxicity tests were performed using MTT assay according to the ISO 10993-5 standard.

[0177] Hydrogels loaded with PeL were cut into discs of 4 or 10mm diameter and put into a 24 well plate. For each condition, three hydrogels were tested in order to have technical replicates. Well plate was sterilized using UV-light for 15-30 min.

[0178] For direct cytotoxicity assays, mouse embryonic fibroblast cell line “Balb 3T3 cells” (ATTC CCL163, passage 5 to 25) were cultured in DMEM medium (DMEM with 10% of FBS and 1 % of penicillin-streptomycin) and incubated at 37°C in Thermo Scientific™ Heracell™ 150i CO2 incubators. About 6x104Balb 3T3 cells / well were seeded in 24 well plates and allowed to attach for 24h (37°C, 5% CO2 atmosphere). Then, 4mm sterile hydrogel discs were added to the monolayer of Balb 3T3 cells (80% of cell confluence). After 24h, hydrogel discs were removed, and MTT test was performed in order to measure cell metabolic activity. For MTT assay, the cells were incubated for 3h in 0.2 mg.mL"1of MTT diluted in cell culture medium. The medium was then removed, and formazan (purple crystals formed as a result of metabolic reduction by viable cells) was dissolved in dimethyl sulfoxide DMSO (0.5 mL of DMSO was added per well).

[0179] For indirect cytotoxicity assays, extraction method was performed. Sterile hydrogels discs of 10mm diameter were placed in 24 well plate and 1.256 mL of cell culture medium (DMEM) was added to each well. The plate was sealed with plastic film to avoid evaporation and incubated at 37°C, 5% CO2 while shaking for24h. About 6.103of Balb 3T3 cells (passage 5 to 25) were seeded in 96 well plate and incubated for 24h at 37°C, 5% CO2 to reach 80% of cells confluence. After 24h, 100 pL of hydrogel extracts were added to the 96 well plate containing the cultured cells. The plate was then sealed and kept at 37°C, 5% CO2 for 24h with mild shaking. After 24h, extraction media is removed and 100 pL of MTT solution previously diluted in DMEM was added to each well. MTT medium was removed after 3h of incubation (37°C, 5% CO2,), and 100pL of dimethyl sulfoxide (DMSO) was added to dissolve formazan salts.

[0180] For biocompatibility assay, the absorbance of each well was measured at 570 nm with a Xenius spectrophotometer (Safas, Monaco). Data were collected, normalized and expressed in percentage of cell viability. Seventy percent of cell viability is considered as the accepted threshold to consider the compound as “non- cytotoxic” according to ISO standard (ISO 10993-5:2009 https: / / www.iso.org / fr / standard / 36406.html).

[0181] Figure 9 shows indirect (9A) and direct (9B) cytotoxicity results of Car hydrogels loaded with PeL polycations evaluated on Balb 3T3 cells using MTT test. Representative microscopy images of Balb 3T3 cells after 24h of incubation are also presented. Results demonstrate that all hydrogels presented more than seventy percent of cell viability; and according to ISO 10993-5 standard, Car loaded hydrogels are not cytotoxic. Moreover, results show that PeL incorporated in hydrogels did not induce cytotoxicity on Balb 3T3 cells at the different concentration used (5 or 7.5 mg / mL).

[0182] RHEOLOGICAL PROPERTIES OF HYDROGELS

[0183] The viscoelastic properties of carrageenan-based hydrogels loaded or not with polycations were measured to assess the viscoelastic properties of hydrogels before and after loading, using a KINEXUS rheometer Ultra+ (Malvern Instruments, United Kingdom) equipped with a plate-plate geometry (20 mm diameter) and a 50 N load cell. Rectangle hydrogel samples with a diameter of 20x20mm and 2mm thickness were used for the rheological tests. Samples were placed in the rheometer plate and were covered to prevent any water evaporation during the experiment. Temperature was equilibrated at 25°C for 5min before testing. The frequency sweeps in the range of 0.01 to 10 Hz. Storage modulus (G’) and loss modulus (G”) were measured as a function of frequency and results were reported as the mean ± standard error of at least 3 specimens.

[0184] The changes in the storage modulus (G’) and loss modulus (G”) of Car hydrogels loaded or not as a function of frequency are presented in figure 10. For all hydrogels, the G’ remains higher than G” over the entire frequency range confirming the typically solid-like nature of these hydrogels, irrespective to other hydrogels composition. In fact, these results suggest that the elasticity proportion is dominant for Carrageenan-based hydrogels loaded or not with polycations. The incorporation of PeL polycations in the polyanions-based hydrogels remarkably increases the G’ values of hydrogels, which indicate that gel network stability increases with the addition of polycations.

[0185] In conclusion, carrageenan hydrogels loaded with polycations are powerful antiviral, antibacterial and antifungal hydrogels with no cytotoxic effect.

[0186] PEL LOADING IN HYDROGELS AND PEL RELEASING FROM CARRAGEENAN-BASED HYDROGELS

[0187] The loading rate of PeL polycation from Car hydrogels were indirectly quantified using a trace protein detection kit for BC Assay Method according to S.q.Rao et al work (LWT - Food Science and Technology 146 (2021) 111422; (BC Assay Protein Quantification Kit, Advion Interchim Scientific). PeL polycation was solubilized in the buffer solution (distilled H2O, Tris / NaCI or DPBS buffer) at different concentrations. In 48 well plate, 225 pl of PeL solution was added to each well containing hydrogels discs of 6mm of diameter and incubated at 37°C while shaking. After 24h or 48h of loading, hydrogel discs and the remaining supernatants were collected. For each condition, three hydrogels were tested in order to have technical replicates. Loading and releasing rates of Car hydrogels were both calculated from a calibration curve drawn for the standards of PeL using BC kit.

[0188] The loading rate was evaluated as follows:

[0189] A volume of 25 pL of the remaining supernatants were put into 96 well plate, and 200 pL of BC assay working reagent was added to each well and heated at 37°C for 30 minutes. Optical absorbance was measured using Thermo Scientific™ Varioskan™ LUX multimode microplate reader at 562 nm.

[0190] The loading rate of Car hydrogels with PeL polycation (% PeL loading) were calculated according to the calculation formulas (1) and (2):

[0191] Loading %(PEL) = (CV - CiVi) x 100 (2) where C, Ci and Cf are the concentration of PeL loaded in the hydrogel discs, the initial concentrations of PeL added to hydrogel discs at to and the final concentrations of PeL in the remaining supernatants, respectively. V, V; and Vf are the volume of hydrogel discs (V=0, 05652 mL), the volume of PeL solution added to hydrogel discs (Vj= 0.225 mL) and the volume of the remaining supernatant (Vf= 0,19 mL), respectively.

[0192] Results of the loading rates of Car hydrogels with PeL expressed in percentages (%) are shown in table 6. The concentrations of PeL in the initial solutions used for loading were 5 or 7.5 mg / mL and results show that hydrogels were loaded with 47 to 90% of the PeL incorporated in the loading solution regardless the initial concentration of PeL used for loading. Table 6. Loading rate of carrageenan hydrogels with PEL using BC kit method.

[0193] CUMULATIVE RELEASED AMOUNT OF POLYLYSINE FROM CARRAGEENAN-BASED HYDROGELS

[0194] The cumulative amount of released polylysine was evaluated as follows:

[0195] After loading of hydrogel discs with PeL, hydrogels were collected into 48 well plate, washed with buffer solution and sterilized by UV-light for 30 minutes. A volume of 300 pl of the buffer solution was added to each well. Hydrogel discs were incubated at 37°C while shaking. The buffer solutions in each well were collected at different time intervals (after 1 , 2, 3, 4, 5, 6 and 7 days) and was then replaced by a fresh one. The cumulative amount of released polylysine was calculated from a calibration curve drawn for the standards of PeL using BC kit.

[0196] Curves for cumulative amount of released polylysine from Car hydrogels at 37°C are shown in figure 11. Results show similar curves trend over time regardless hydrogels composition. The released PeL presented a significant burst effect at the first day, then, PeL was gradually released over time to finally reach stability.

Claims

CLAIMS1. A composition comprising (poly)lysine and a mixture of A-carrageenan and K-carrageenan.

2. The composition of claim 1 or 2, wherein the (poly)lysine is selected from the group consisting of: a-poly-L-lysine, e-poly-L-lysine, a-poly-D-lysine, e-poly-D- lysine, and mixtures thereof.

3. The composition of any one of the preceding claims, wherein the (poly)lysine has the following formula (1):n being an integer comprised from 10 to 100, preferably from 10 to 30.

4. The composition of any one of the preceding claims, comprising from 20% to 65%, preferably from 30% to 60%, and more preferably from 35 to 55% by weight of (poly)lysine in relation to the total weight of said composition.

5. The composition of any one of the preceding claims, wherein the ratio between the weight of A-carrageenan and the weight of K-carrageenan is from 1 / 20 to 1 / 1 , and is preferably 1.

6. The composition of any one of the preceding claims, comprising from 1% to 4%, preferably from 1% to 2%, by weight of K-carrageenan in relation to the total volume of said composition.

7. The composition of any one of the preceding claims, comprising from 1% to 4%, preferably from 1% to 2%, by weight of A-carrageenan in relation to the total volume of said composition.

8. The composition of any one of the preceding claims, comprising from 0.05% to 5%, preferably from 1% to 3%, more preferably from 1.5% to 2%, by weightof the mixture of A-carrageenan and K-carrageenan in relation to the total volume of said composition.

9. The composition of any one of the preceding claims, wherein the A- carrageenan and the K-carrageenan have a molecular weight greater than 200 kDa.

10. The composition of any one of the preceding claims, wherein the mixture of A-carrageenan and K-carrageenan is in the form of a hydrogel.

11. The composition of any one of the preceding claims, being in the form of a hydrogel.

12. A device comprising a coating comprising a composition according to any one of claims 1 to 11.

13. The composition of any one of claims 1 to 11 for use for the prevention of viral infections and / or bacterial infections and / or fungal infections.

14. The use of the composition of any one of claims 1 to 11 in a cosmetic composition, as a coating, as a disinfectant, as means for sexual protection, or as a cleaning solution for lenses.

15. The use of the composition of any one of claims 1 to 11 as an antiviral agent and / or as an antibacterial agent and / or as an antifungal agent.

16. The composition of any one of claims 1 to 11 for the prevention of sexually transmitted infections.

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