Novel formulations for biobased nanostructured films and coatings

Abstract

The present invention refers to novel compositions based on deacetylated chitin nanowhiskers (CsNWs) as well as to processes for their preparation. Furthermore, the invention relates to a process for preparing films or coatings starting from said compositions as well as to biomedical, cosmetic articles based upon said compositions and uses thereof.

Description

[0001] Novel formulations for biobased nanostructured films and coatings

[0002] The present invention relates to novel compositions based on deacetylated chitin nanowhiskers (CsNWs) as well as to processes for their preparation. Furthermore, the invention relates to a process for preparing films or coatings starting from said compositions as well as to biomedical, cosmetic articles based upon said compositions and uses thereof.

[0003] STATE OF THE ART

[0004] Chitin is the second most abundant biopolymer in nature and is a polysaccharide composed of N-acetyl-glucosamine units linked together with p-1 ,4-type linkages leading to the formation of a highly crystalline polymer. Chitin exists in three different polymeric forms; the a form is the most common and is easily obtainable as waste from the fishing industry which produces 6-8 million tons of shells between shrimps, crabs, and lobsters, of which 14-40% is a chitin. From the partial deacetylation of chitin, it is possible to obtain chitosan, less present in nature, but more easily soluble (e.g., in slightly acidified water). Various functional properties of chitin and chitosan are known such as biodegradability, biocompatibility, non-toxicity, absence of odor, adsorbent properties, antibacterial and anti-inflammatory activity.

[0005] Interestingly, the use of chitin and chitosan in nanostructured form, with reference to nanofibrils, also known as nanowhiskers (NWs), whether made of chitin (CtNWs) or deacetylated on the surface (CsNWs), is gaining increasing interest over the last years. Indeed, the nanometric conformation of these materials allows not only to maintain the properties listed above, but also to amplify them thanks to the increase in the surface area and volume ratio.

[0006] For the above reasons, NWs have been used for example as fillers within polymeric matrices of both synthetic or natural origin, such as starch, polyvinyl alcohol (P A), chitosan, carrageenan, and PBS (polybutylene succinate), mainly aimed at improving the mechanical properties that characterize the starting polymer. Furthermore, thanks to their antimicrobial and gas barrier properties, NWs have been used in the production of active materials to be applied in food packaging. Composites produced with chitin and derivatives have also found application in the medical sector due to the ability, supported by in vitro and in vivo clinical tests, to accelerate wound healing. The known antimicrobial properties conferred by the positive charges more present in chitosan than chitin also allow to minimize the possibility of wound infections by bacteria, viruses, and fungi. Biocompatibility and non-toxicity make it possible to use NWs as a vehicle for the transport of drugs (drug delivery), as a scaffold in the field of tissue engineering and in the formulation of products for personal care. Said capabilities have been also exploited in different fields such as agriculture, where chitin and chitosan, at the nanometric scale, have been the subject of several studies as adjuvant agents for plant growth to replace the use of agrochemical products with a greater impact on human health and of the environment. For example, they have been reported to reduce germination times and improve seedling growth yield by influencing photosynthesis and therefore chlorophyll content and nutrient uptake.

[0007] Despite the numerous progresses made in the use of chitin and chitosan at the nanometric scale, there is however still a need for the development of efficient protocols for the production of functional materials made of such compounds, such as films or coatings, which could conjugate optimized mechanical-physical properties with greater versatility and scalability at an industrial level.

[0008] SUMMARY OF THE INVENTION

[0009] The technical problem posed and solved by the present invention is that of providing optimized compositions based on CsNWs, which are particularly suitable for the fabrication of functional materials such as films or coatings, and whose properties can be properly tuned for a plethora of different applications, ranging from the production of biomedical or cosmetic articles up to the fabrication of packaging materials and preservation of paper and ancient manuscripts.

[0010] The authors of the invention have indeed developed versatile aqueous compositions based on CsNWs, which can be used to produce free-standing films as well as coatings suitable for applications by means of an easy and scalable procedure. To this end, the compositions according to the present invention particularly comprise a combination of CsNWs with an acidifying agent, a plasticizer, and an aqueous solvent. Such combination permits to produce films or coatings containing CsNWs, which, compared to the products of the prior art, are able to conjugate the antibacterial and antifungal properties of the NWs with excellent barrier properties against gas permeation as well as the desired optimal flexibility.

[0011] Advantageously, the compositions may further comprise at least one polyphenolic compound, whose presence has been found particularly useful to impart the compositions and resulting films or coatings with improved antioxidant, antibacterial, and anti-UV properties. According to the invention, further additives may be incorporated in the compositions, such as conductive compounds or even specific active ingredients or drugs, which can be used to increase the mechanical properties of the resulting materials or to impart them with further properties of interest for the desired application.

[0012] Compared to the formulations already known in the art, the compositions of the invention have the advantage of being 100% natural and biodegradable as well as presenting, as already underlined, a high degree of versatility, permitting indeed the production of films or coatings substantially consisting of nanowhiskers.

[0013] Thanks to the specific combination of technical features that will be disclosed in the present description and in the claims, the compositions and processes developed by the authors of the invention offer important advantages.

[0014] Notably, as clearly demonstrated by the studies of direct and indirect cytotoxicity on fibroblasts reported in the experimental section of the present specification, the compositions, as well as the films obtained from such compositions developed by the authors of the invention can exhibit good biocompatibility and hemocompatibility, as confirmed for example by the variations of the prothrombin time (PT) and activated partial thromboplastin time (aPTT). As a consequence, the films and coatings that can be produced starting from said compositions are suitable both for dermal contact and for implantation inside the body (e.g., in the form of a stent).

[0015] The high versatility of the invention, which can be tuned as mentioned also based on the nature and amount of the polyphenol additive contained in the composition, is also confirmed by the platelet aggregation data reported in the experimental section. These data demonstrate that the antithrombotic properties of the composition can be easily modulated by varying the quantity and type of polyphenol used. In such a way, it is possible to go from films or coatings that favor platelet adhesion to materials that show complete platelet anti-adhesion activity, with results therefore similar to the use of heparin, a drug widely used as an anticoagulant. This characteristic, together with the well-known antimicrobial and antioxidant properties of chitosan and polyphenols, allows to formulate films for external and internal use with antithrombotic properties as well as for protection of the wound from the risk of infection.

[0016] In the light of the above, the inventors have found that the compositions of the invention are particularly suitable for application in the medical or cosmetic sector, for example to produce protective patches in the form of films. Alternatively, the compositions can be directly applied in the form of sprays onto the skin of a subject for the treatment of wounds, lesions, burns and abrasions or else for the development of stents useful to avoid the onset of post-operative complications.

[0017] In one embodiment, thanks to the combination with suitable additives as will be further illustrated below, the compositions of the present invention can be in the form of a nail polish useful for promoting reinforcement of the nails or for treatment or prevention of onychomycosis and / or onychophagies. Advantageously, compared to formulations known in the art, the nail polish comprising any of the compositions according to the present invention, once dry, does not undergo any washout, allowing the operator not to give up his / her daily hygiene.

[0018] The same compositions, combined with natural additives which stimulate the growth and strengthening of the nail (i.e. , vitamin H, provitamin B5), can be used as a medical device for the formulation of repair polishes for the treatment of traumatized, weakened, scratched and brittle nails. By adding an additive that gives a bitter taste (i.e., denatonium benzoate), the nail polish of the invention can be used to fight nail biting by exploiting the dual action of discouraging gnawing and strengthening and nourishing the nail. The application by brush or spray allows to form a completely transparent, non-glossy and odorless film that can also be applied to the surrounding cuticles, meeting the needs of individuals of all sexes and ages. Furthermore, the antimicrobial character of chitosan allows to protect damaged nails and cuticles from possible infections. The proposed compositions also represent a valid alternative to the water-based covering cosmetic nail polishes available on the market, which often contain non-naturally derived filming agents such as acrylates.

[0019] Thanks to the proper combination of its components, the compositions according to the present invention can be further used to produce economic, biodegradable, natural-origin active / intelligent packaging that does not alter in any way the food with which it comes into contact. Such packaging is not only suitable for food contact (chitosan is already recognized by GRAS as safe) but falls into the category of edible films for active packaging. The addition of a wide range of additives of natural origin allows, as already mentioned, to modulate the properties of the NWs- based film by implementing its functional properties and at the same time maintaining the characteristics of biodegradability and edibility.

[0020] In addition to the above, the compositions according to the present invention can be employed to produce biocompatible and biodegradable conductive films for biological applications and in the sensing sector.

[0021] Furthermore, said compositions can be effectively used to produce functional films or coatings that can act as fungicide, antifungal, plant growth aid, defense system stimulant materials in the field of agriculture, as well as an antiperspirant agent to reduce water consumption. With this regard, CsNWs-based films can indeed act by increasing reflectance, thus lowering leaf temperature, and reducing evaporation, and on the other hand as a physiological regulator of the stomata by influencing abscisic acid (ABA), which is a phytohormone that regulates, among other things, the consumption of water. The formation of the films on leaves and fruits can advantageously vary the reflected component of light, making everything less attractive to insects and parasites and therefore acting as a natural repellent. The addition of polyphenols to the compositions (e.g., lignins and tannins) amplify the range of protection against UV rays without hindering the photosynthetic processes, while providing, as already mentioned, suitable antimicrobial and antifungal properties.

[0022] Compared to other photoprotectors already on the market, the compositions based on CsNWs could allow to bypass the leaching step of the film, which is normally needed before placing the products on the market and takes place through mechanical rinsing or by preventive interruption of the treatment in the period before the harvest. In fact, the presence of the almost completely transparent CsNWs films not only does not alter the palatability of the harvest products in any way, but also acts as an active packaging, preserving freshness and increasing their shelf life.

[0023] In addition to the above, the inventors have found that the compositions according to the present invention can be advantageously used for the preparation of coatings for preventing or minimizing degradation of paper, in particular ancient manuscripts, documents, or photographs, which is mainly caused by physical (temperature, light, humidity) and biological agents (insects, bacteria, and molds). As will be clearly explained in the experimental section of the present specification, the formulation of a nanostructured chitin-derived multifunctional coating is indeed capable of counteracting the different degradative processes thanks to the numerous properties of the starting material.

[0024] It forms hence part of the present invention:

[0025] - A composition comprising deacetylated chitin nanowhiskers (CsNWs), an acidifying agent, and an aqueous solvent; preferably wherein said composition further comprises a plasticizer;

[0026] - A process for preparing a composition according to any of the embodiments disclosed herein, comprising at least the following steps:

[0027] (i) providing a suspension of deacetylated chitin nanowhiskers (CsNWs);

[0028] (ii) dispersing said suspension into an aqueous solvent;

[0029] (iii) adding an acidifying agent to the dispersed suspension;

[0030] (iv) preferably adding, in any one of the above-mentioned steps, a plasticizer;

[0031] - A process for preparing a film or coating comprising deacetylated chitin nanowhiskers (CsNWs), said process comprising the following steps:

[0032] (i) applying a composition according to any one of the embodiments disclosed in the present specification onto a target surface or substrate; and

[0033] (ii) allowing the aqueous solvent of said composition to evaporate from the composition so as to form said film or coating.

[0034] - A film or coating obtainable by a process according to any one of the embodiments disclosed herein.

[0035] - A biomedical, sanitary, or cosmetic article comprising or consisting in a composition or in film or coating according to any of the embodiments disclosed in the present specification, said film or coating being optionally deposited or adsorbed onto a support.

[0036] - A composition according to any of the embodiments disclosed herein for use as a medicament.

[0037] - A use of a composition according to any of the embodiments disclosed in the present specification for preventing or minimizing degradation of paper, preferably of ancient manuscripts, documents, or photographs.

[0038] - A process for the fabrication of a film or coating onto a substrate for packaging of drugs, food and / or beverages, wherein said fabrication is carried out by means of a process according to any one the embodiments disclosed herein.

[0039] - A process for the fabrication of a sensor or biosensor, comprising a step of forming a film or coating onto a substrate, wherein said fabrication is carried out by means of a process according to any one of the embodiments disclosed herein.

[0040] Other advantages of the invention will be apparent from the following detailed description of preferred embodiments of the invention.

[0041] DETAILED DESCRIPTION OF THE FIGURES

[0042] Figure 1. A) Suspension of CsNWs in water before the addition of the acidic solution; B) Colloidal suspension of CsNWs after the addition of the acidic solution; C) CsNWs-based film formed after the complete evaporation of the solvent.

[0043] Figure 2. A) Atomic force microscopy (AFM) micrograph of CsNWs; B) Number size distribution of CsNWS as measured by dynamic light scattering (DLS); C) Number size distribution of CtNWs as measured by DLS.

[0044] Figure 3. A) Fourier-transform infrared spectroscopy (FT-IR) spectra of CtNWs and CsNWs,

[0045] B) Thermogravimetric analysis (TGA) curves of CtNWs and CsNWs (under N2 atmosphere). Figure 4. CsNWs-based films: A) film casted in a Teflon mould (the mould has been produced by a computer numerical control milling machine; the mould pattern has been reproduced on the film); B) film casted in a smooth silicone mould.

[0046] Figure 5. A) Photograph of a CsNWs-G film; B) Photograph of a bended CsNWs-G film; C) Scanning electron microscopy (SEM) micrograph of the surface of a CsNWs-G film.

[0047] Figure 6. A) water drops deposited on the surface of CsNWs-based films for water contact angle measurements; B) Variation of the volume of the water drop deposited on the film surface over 300 s; C) Film weight increase due to water absorption after 10 min of incubation; D) Film thickness increase due to water absorption after 10 min of incubation E) Photographs of CsNWs-based films before and after incubation in water.

[0048] Figure 7. Cartoon showing the interactions of the different polyphenolic additives with the CsNWs.

[0049] Figure 8. Representative stress-strain curves of the CsNWs-based films: A) formulations containing 1 wt% of polyphenols as compared to the CsNWS-G sample; B) formulations containing different concentrations of methanol insoluble hardwood lignosulfonate fraction (MIHLS).

[0050] Figure 9. Amount of polyphenols released in water as a function of the incubation time: A) formulations containing 1 wt% of polyphenols as compared to the CsNWS-G sample; B) formulations containing different concentrations of MIHLS.

[0051] Figure 10. Indirect contact test. A) Schematic representation of the indirect test. B) NIH-3T3 cell viability results evaluated at 24 hours after incubation without (control, Ctrl) or with increasing serial dilutions of the extracts collected after 24 hours from the films. The results of the viability test were expressed as a percentage compared to the viability of the NIH-3T3 cells on Ctrl set at 100%.

[0052] Figure 11. Direct-contact test. A) Schematic representation of direct-contact test. B) NIH-3T3 cell viability results assessed at 24h from the seeding on the different film. Results of viability test were expressed as a percentage related to NIH-3T3 cell viability on tissue culture plates (TOPS) set as 100%. *Significant differences at p<0.05. C) Representative confocal laser scanning microscopy (CLSM) images (scale bar: 50 pm) of cell adhesion after 24 h of culture on films. Focal adhesion protein pi integrin expression in green (Alexa Fluor 488) and nuclei in blue (Hoechst 33342). Figure 12. Direct contact test. Representative optical images of cells seeded and cultured for 24 hours on different films. Different fields were shown for each type of sample.

[0053] Figure 13. PT (A) and APTT (B) values of the platelet poor plasma (PPP) in contact with the different films. Ctrl represents PPP incubated in test tubes without any material present.

[0054] Figure 14. Human fibrinogen (hFg) absorption on the different films. A) hFg absorption expressed as pg / cm2. B) hFg absorption expressed as percentage related to Ctrl set as 100%. Ctrl represents hFg absorption on high binding well plates. *Significant differences at p<0.05.

[0055] Figure 15. Adhesion of human platelets on different films. A) Determination of human platelet adhesion by lactate dehydrogenase assay (LDH) assay as described in the Materials and methods section. *Significant differences at p<0.05. B) Representative CLSM images of human platelets stained with phalloidin-TRIC (in red) on the different films. The positive control (Ctrl+) represents human platelet adhesion on the collagen l-coated well, while the negative control (Ctrl ) represents adhesion on the bovine serum albumin (BSA)-coated well (scale bar: 20 pm).

[0056] Figure16. Optimized process to produce the CsNWs-based formulation and photograph of a coated paper sheet.

[0057] Figure 17. Coated paper sheets: A) a flower drawing can’t be removed after the application of the coating; B) a methylene blue solution does not permeate the coated paper sheet.

[0058] Figure 18. A) water drops deposited on the surface of coated and uncoated paper for water contact angle measurements; B) Variation of the volume of the water drop deposited on the coated and uncoated surface over 150 seconds.

[0059] Figure 19. Comparison between the formulation with 100% H2O, H2O / ETOH= 3, and H2O / ETOH= 2. Red X: coating removed. Yellow X: coating partially removed, Green X: polish can’t be removed.

[0060] Figure 20. Photographs of fingernails after the application of the CsNWs-based polish: A) uncolored formulation; B) formulation containing Rose Bengal, C) formulation containing Indigo Carmine.

[0061] GLOSSARY In the context of the present specification, the term “nanowiskers”, herein abbreviated also as NWs, is intended to mean one-dimensional nano-elements with a width or diameter (or, generally, a cross-dimension) of nanometer size. Chitin or deacetylated chitin nanowhiskers are also referred to in the art as "nanofibrils" or, in this context, simply as "fibrils", and such terminology, as used in the present application, is equivalent to the term "nanowhiskers". In preferred embodiments of the invention, average nanowhisker lengths typically do not exceed 400 nm and more preferably are about 250-300 nm. The average length of the nanowhiskers prepared according to the present invention can be estimated using any of the techniques known by the skilled person, for example by means of dynamic light scattering (DLS) in a pH range wherein the suspensions are stable (e.g., between 3-6.5 pH).

[0062] As used herein with reference to a film according to any of the embodiments disclosed in the present specification and in the claims, the terms “self-standing” or “free-standing” means that the film can be in the form of a sheet and can be handled as an independent sheet without the assistance of a substrate or support. Thus, the term "self-standing" may have the same meaning as "self- supporting".

[0063] In the context of the present specification, the expressions “deacetylated chitin nanowhiskers” and “chitosan nanowhiskers” can be interchangeably used and are herein abbreviated as CsNWs.

[0064] In the context of the present description, the term “never-dried gel” refers to an aqueous dispersion or suspension of chitin or chitosan NWs and, in particular, deacetylated chitosan NWs, which are obtained after processing from their various sources according to any of the processes disclosed in the present specification and never undergo any dehydration process.

[0065] As used herein, the term "lignin" has its normal connotation in the art and refers to the class of complex organic polymers made by cross-linking phenolic precursors that form key structural materials in the support tissues of most plant. As used herein, the term "lignosulfonate" refers to the reaction product of lignin which is inherently obtained during the sulfite pulping of wood, straw, corn stalks, bagasse, and the like, and is a principal constituent of the spent sulfite liquor which is derived from that process.

[0066] As used herein, the term "free-standing film" refers to a film capable of maintaining the shape of a film by itself without a separate support at room temperature and atmospheric pressure.

[0067] In the context of the present description, “about” refers to the experimental error that can occur during conventional measurements. More particularly, when referring to a value it indicates ± 5% of the indicated value.

[0068] Anywhere in the present description and in the claims, the word “comprising” may be replaced by the word “consisting of”.

[0069] DETAILED DESCRIPTION OF THE INVENTION

[0070] Compositions

[0071] As previously mentioned, a first object of the present invention is represented by a composition comprising deacetylated chitin nanowhiskers (CsNWs), an acidifying agent, an aqueous solvent. According to a preferred aspect, the composition according to any of the embodiments disclosed in the present specification and in the claims further comprises a plasticizer.

[0072] Deacetylated chitin NWs according to the invention can be obtained by surface deacetylation of chitin NWs (CtNWs) by means of any of the procedures known to a person skilled in the art.

[0073] In one aspect, CsNWs are prepared by surface deacetylation of a suspension of CtNWs with an inorganic base such as sodium hydroxide (NaOH), under reflux and mechanical stirring. Preferably, stirring is carried out for a period not exceeding 12 hours, since it has been found that longer reaction times can result in a degradation of the polysaccharidic chains, thus providing a dark grey / brown product.

[0074] Merely by way of example, CsNWs can be prepared by surface deacetylation of a never- dried gel of CtNWs using a solution of 12.5 N NaOH under reflux and mechanical stirring for 12 hours, at a CtNWs / NaOH solution ratio of 60 g / mL. Preferably, the suspension of CsNWs resulting from surface deacetylation according to any of the processes disclosed herein is left to cool at room temperature before subsequent processing. In one aspect, the suspension of CsNWs resulting from surface deacetylation is subjected to a number of washing steps and centrifugation cycles until a neutral pH is reached. Washing is preferably performed using water, in particular deionized water.

[0075] The starting chitin material used to prepare CtNWs and / or CsNWs according to any of the variants disclosed herein can be obtained from various sources known to a person skilled in the art, preferably is purified chitin from crab or shrimp shells. Merely by way of example, chitin can be obtained from shrimp shells by means of a purification step comprising a basic treatment followed by a bleaching step to remove residual proteins and inorganic impurities.

[0076] In one aspect, CtNWs that are subjected to deacetylation to produce CsNWs can be prepared according to any of the procedures known in the art. Among the multiple methods of obtaining chitin NWs, the most widely used is acid hydrolysis which allows to extract the crystalline component of the polymer characterized by high mechanical properties.

[0077] In one aspect, CsNWs are obtained by deacetylation of CtNWs that are prepared by means of hydrolysis of chitin, for example chitin in the form of flakes or powder, in the presence of an inorganic acid under stirring. Preferably, hydrolysis of chitin flakes is carried out with a solution of hydrochloric acid (HCI) 3N under reflux and mechanical stirring for 4 hours. More preferably, a chitin / HCI solution ratio of 30 g / mL is used in such process. After hydrolysis of the chitin flakes is carried out according to any of the above procedures, the resulting mixture is allowed to cool to room temperature. Subsequently, the resulting mixture can be subjected to a number of washing steps, preferably in deionized water, and centrifugation cycles. Preferably, the precipitate recovered after centrifugation is re-suspended into an aqueous solvent, preferably water, and subjected to a sonication step, in particular by means of horn-tip sonication.

[0078] Following sonication, the resulting suspension can be diluted with an aqueous solvent, preferably water, and treated with a base, such as NaOH, until a complete precipitation of the CtNWs is achieved. The result is a suspension or never-dried gel of CtNWs which can be then subjected to a number of washing steps and centrifugation cycles until a neutral pH is reached. This last step is particularly important to remove residual salts. Preferably, the speed of centrifugation is progressively increased at each of the above cycles to obtain a better precipitation of the CtNWs and a more compact gel of CtNWs, hence contrasting the pH decrease.

[0079] In one aspect, CsNWs of the composition of the invention are deacetylated to a degree (i.e. degree of deacetylation, herein abbreviated as DA) of 30% or more, still preferably 30 to 95% and most preferably 35 to 70%. In various embodiments, the DA of the CsNWs is however not more than 40%.

[0080] The DA may be readily controlled by appropriately selecting the concentration of the alkali to be used in the deacetylation process, as well as the treating temperature, and the treating time. As the skilled person knows, the DA of chitin NWs can be determined by elemental analysis, in particular by means of a CH NS analyzer, as illustrated for example in the experimental section of the present specification.

[0081] The content of CsNWs in the compositions of the invention would vary depending on the final application or dosage forms. As will be further explained in the following sections, the amount of CsNWs in the compositions of the invention can be properly adjusted by the skilled person in the art depending on the desired properties of the film or coating that can be prepared starting from such composition, for example depending on the desired size and / or thickness of the film or coating.

[0082] The amount of CsNWs or CtNWs in any of the compositions / suspensions according to the present invention can be determined by means of any of the methodologies known to a skilled in the art, preferably by means of gravimetric analysis after drying the sample at 100°C until complete evaporation of the aqueous solvent.

[0083] In one preferred aspect, the composition according to the invention comprises CsNWs in an amount of between 1 and 100 mg per mL of the composition, more preferably between 20 and 70 mg / mL, even more preferably equal to 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg per mL of the composition, even more preferably equal to 25 mg / mL or 35 mg / mL. For applications requiring a higher density of CsNWs in the composition, an amount of 50 mg / mL of CsNWs is preferred.

[0084] According to the invention, the addition of an acidifying agent to the compositions promotes dispersion of said CsNWs in the aqueous solvent. Any suitable organic or inorganic acidifying agent known in the art can be added to the composition to such purpose.

[0085] In one preferred aspect, the composition according to the present invention comprises an acidifying agent selected from acetic acid, lactic acid, azelaic acid and HCI. Preferably, the composition comprises acetic acid or lactic acid as acidifying agent.

[0086] Given the presence of the acidifying agent, the composition according to any of the embodiments disclosed herein is an acidic composition, preferably is a composition having a pH between 4 and 6.5, more preferably between 5.4 and 6.5, even more preferably between 5.4 and 5.6 or between 5.8 and 6.1. In one aspect, the acidifying agent is hence added to or is present in a composition according to any of the embodiments disclosed herein in an amount suitable to provide an acidic pH, preferably a pH comprised between 4 and 6.5.

[0087] According to the invention, the plasticizer that is preferably present in the composition can be any known plasticizing agents. Preferably, said plasticizer is a plasticizing agent of natural origin.

[0088] As will be explained in detail in the following paragraphs as well as shown in the experimental section of the present specification, the presence of a plasticizer in the compositions according to the present invention allows to modulate the flexibility of a film or coating that can be produced starting from such compositions.

[0089] A particularly suitable plasticizer that can be present into any of the compositions according to the present invention is selected from the group consisting of glycerol, triethyl citrate, polyethylene glycol (PEG), glycerol oligomers, for instance diglycerol, and combinations or mixtures thereof. Preferably, the plasticizer is at least one polyol or a mixture of polyols, more preferably glycerol.

[0090] In one aspect, said plasticizer is preferably present in an amount equal to 20% or more in the composition, more preferably 30% or more by weight based on the total weight of the CsNWs in the composition. Advantageously, said plasticizer can represent 2% to 50%, preferably 10% to 30%, most preferably 30% by weight based on the weight of the CsNWs in the composition. At such contents said plasticizer improves flexibility of the films or coatings that can be produced starting from said compositions.

[0091] The aqueous solvent present in a composition according to any of the embodiments disclosed herein can be any known aqueous solvent, preferably is water or a mixture of water with an alcohol, more preferably a mixture of water and ethanol (EtOH). In one aspect, the composition according to the invention comprises water or a mixture of water and alcohol at a water / alcohol ratio of between 1 and 3 volume / volume (v / v), preferably at a ratio equal to 1 , 2, or 3 v / v.

[0092] In one preferred aspect, a composition according to any of the embodiments disclosed herein further comprises at least one polyphenolic compound. A suitable polyphenolic compound is any of the polyphenolic compounds known in the art, in particular a polyphenolic compound selected from tannins, such as condensate, hydrolyzed and / or monomeric tannins, lignin, or derivatives thereof. Merely by way of example, derivatives of lignin include lignin fractions, sulfonate lignin, lignosulphonates, as well as lignin nanoparticles or microcapsules, which can optionally encapsulate one or more active ingredients. Any type of lignin that is hardwood, softwood, grass, crude / technical, or pure may be employed.

[0093] In one preferred aspect, the composition comprises at least one polyphenolic compound selected from tannic acid (TAN), epigallocatechin gallate (EPCG), mimosa tannin (MIM), lignosulfonates such as hardwood lignosulfonates (HLS), in particular methanol insoluble hardwood lignosulfonate fractions (MIHLS), Kraft lignin (KL), in particular ethanol insoluble Kraft lignin fraction, lignin nanoparticles or combinations thereof.

[0094] In one aspect, said at least one polyphenolic compound is present in the composition in an amount of between 1 % to 40% by weight with respect to the total weight of CsNWs in the composition, more preferably between 1-20%, even more preferably equal to 1%, 2%, 2.5%, 5%, 10%, 15% or 20%. At such contents said at least one polyphenolic compound improves the antioxidant, antibacterial and anti-UV properties of the composition as well as films and coatings that can be obtained from such composition.

[0095] Preferably, when said at least one polyphenolic compound comprises tannins, these are present in an amount of between 1 and 3% by weight with respect to the total weight of CsNWs in the composition.

[0096] Preferably, when said at least one polyphenolic compound comprises lignins, these are present in an amount of between 1 and 20% by weight with respect to the total weight of CsNWs in the composition. In one aspect, said at least one polyphenolic compound is tannic acid and is present in the composition in an amount of between 1 % to 20% by weight with respect to the total weight of CsNWs in the composition, more preferably equal to 1%, 2.5%, 5%, 10% or 20%.

[0097] In another aspect, said at least one polyphenolic compound is epigallocatechin gallate and is present in the composition in an amount of between 1 % to 5% by weight with respect to the total weight of CsNWs in the composition, more preferably equal to 1%, 2.5%, or 5%.

[0098] In another aspect, said at least one polyphenolic compound is mimosa tannin and is present in the composition in an amount of between 5% to 15% by weight with respect to the total weight of CsNWs in the composition, more preferably equal to 5%, 10%, or 15%.

[0099] In another aspect, said at least one polyphenolic compound is hardwood lignosulphonate and is present in the composition in an amount of between 1 % to 20% by weight with respect to the total weight of CsNWs in the composition, more preferably equal to 1 %, 10%, or 20%.

[0100] In another aspect, said at least one polyphenolic compound is a methanol insoluble hardwood lignosulphonate fraction and is present in the composition in an amount of between 5% to 20% by weight with respect to the total weight of CsNWs in the composition, more preferably equal to 5%, 10%, or 20%.

[0101] In another aspect, said at least one polyphenolic compound is an ethanol soluble Kraft lignin fraction and is present in the composition in an amount of between 2.5% to 5% by weight with respect to the total weight of CsNWs in the composition, more preferably equal to 2.5%, or 5%.

[0102] In another aspect, said at least one polyphenolic compound comprises nanoparticles from acetone soluble lignin fraction or from enzymatically oxidized acetone soluble lignin fraction and is present in the composition in an amount equal to 10% by weight with respect to the total weight of CsNWs in the composition.

[0103] The composition according to any of the embodiments disclosed herein can further comprise at least one conductive compound, in particular a compound selected from biochar, graphene, reduced graphene oxide, carbon dots or any type of conductive nanoparticle known in the art. Preferably, conductive biochar and / or quantum dots obtained as a solid residue from hydrothermal treatment of beer spent grain and, optionally, yeast slurry can be employed. According to another aspect, the composition of any of the embodiments disclosed in the present specification and in the claims comprises at least one pigment or chromophore known in the art. This can be advantageous to impart a desired color appearance to a film or coating that can be prepared from said composition. Preferred pigments or chromophores are represented by indigo carmine, rosa bengala, zinc oxide, and combinations thereof. Alternatively, any fluorophores or luminescent compounds known in the art can be used to such purpose. Preferably, said pigment or chromophore is a biocompatible pigment or chromophore or is a compound of natural origin. One aspect of the present invention particularly refers to a composition having the quali- and / or quantitative formulation as reported in the following tables: Table 1 - Composition a-1)

[0104] Table 2 - Composition a-2)

[0105] Table 3 - Composition b-1)

[0106] Table 4 - Composition b-2) Table 5 - Composition b-3)

[0107] Table 6 - Composition b-4) Table 7 - Composition b-5)

[0108] Table 8 - Composition c-1)

[0109] Table 9 - Composition c-2)

[0110] Table 10 - Composition c-3) Table 11 - Composition d-1)

[0111] Table 12 - Composition d-2)

[0112] Table 13 - Composition d-3)

[0113] Table 14 - Composition e-1)

[0114] Table 15 - Composition e-2)

[0115] Table 16 - Composition e-3) Table 17 - Composition f-1)

[0116] Table 18 - Composition f-2)

[0117] Table 19 - Composition f-3)

[0118] Table 20 - Composition g) Table 21 - Composition h) Table 22 - Composition i)

[0119] Table 23 - Composition j) Table 24 - Composition k)

[0120] Table 25 - Composition I)

[0121] Table 26 - Composition m-1) Table 27 - Composition m-2)

[0122] Table 28 - Composition m-3)

[0123] As will be further explained below, according to the invention, the compositions m-1 , m-2 and m-3 are particularly suitable for use as nail polish compositions.

[0124] According to another aspect, the composition of any of the embodiments disclosed in the present specification and in the claims further comprises at least one compound having antimicrobial properties, in particular a photodegradable compound having antimicrobial properties, and also acting as activity indicator such as methylene blue (herein abbreviated also as MB). As will be further explained below, by virtue of the antimicrobial properties of the CsNWs and the added molecule, such composition is particularly suitable for biomedical applications wherein it is desired to impart antimicrobial properties to a specific substrate.

[0125] In certain aspects, the compositions according to any of the embodiments disclosed herein can further comprise additional excipients or carriers, such as pH regulators, preservatives, buffers, salts, antimicrobial agents, or combinations thereof.

[0126] Depending on the final application, the compositions according to any of the embodiments disclosed herein can further comprise one or more active ingredients such as antibiotics, drugs, antibacterial, antifungal, and / or antimycotic agents, vitamins, antioxidants, or any other agent suitable for treatment or prevention of a particular condition or disease, as well as combinations thereof. The compositions according to any of the embodiments disclosed herein are preferably in the form of a suspension, spray, polish, or paint.

[0127] In one aspect, the compositions according to any of the embodiments disclosed herein are colloidal suspensions, preferably they are stable colloidal suspensions wherein with the term “stable” is herein referred to compositions exhibiting zeta-potential values in the range between ±40 and ±60 mV as measured according to any of the procedures known in the art.

[0128] In one preferred aspect, the acidifying agent, aqueous solvent, the plasticizer and, optionally any additional additives of the composition of the invention are selected among any of the variants disclosed in the present specification and in the claims to provide a composition that comprises or consists of sole biocompatible and / or biodegradable components, i.e. , is 100% biocompatible and / or biodegradable.

[0129] In a particularly preferred embodiment, the compositions according to any of the variants herein disclosed or claimed do not comprise any compound(s) or precursor(s) other than the deacetylated chitin nanowhiskers, which is(are) capable of forming a supporting matrix, in particular it does not comprise any gel precursor(s) (such as alginate), polymers or fillers.

[0130] Processes for preparation of the compositions

[0131] As mentioned in the summary, another aspect of the present invention relates to a process for preparing a composition according to any of the embodiments disclosed in the present specification, which process comprises at least the following steps:

[0132] (i) providing a suspension of deacetylated chitin nanowhiskers (CsNWs);

[0133] (ii) dispersing said suspension into an aqueous solvent;

[0134] (iii) adding an acidifying agent to the suspension dispersed in step (ii), and

[0135] (iv) preferably adding, in any one of the above-mentioned steps, a plasticizer according to any of the variant disclosed in the present specification. Step (i) of the above process can specifically comprise subjecting a suspension of CtNWs to surface deacetylation according to any of the known procedures in the art, such as the ones disclosed in the present specification. In one particular aspect, step (i) comprises subjecting a suspension of CtNWs to surface deacetylation by means of any of the procedures disclosed in the present specification, for example with NaOH under reflux and mechanical stirring.

[0136] It is again preferred that mechanical stirring is performed for 12 hours, more preferably for periods no longer than 12 hours.

[0137] As already mentioned, following deacetylation, it is also preferred that the resulting suspension is subjected to a number of washing and centrifugation cycles until a neutral pH is reached. Each washing step is preferably conducted using deionized water.

[0138] The starting concentration or amount of CsNWs in the suspension provided in step (i) is such that, after dispersion of the suspension in a suitable aqueous solvent in step (ii) followed by acidification in step (iii), a final composition is obtained having an amount of CsNWs according to any of the embodiments disclosed in the present specification and in the claims, preferably comprised between 1 and 100 mg / mL of the composition, more preferably comprised between 20 and 70 mg / mL of the composition, even more preferably equal to 20, 25, 30, 35, 40, 45, 50 mg / mL.

[0139] In one aspect, in said step (ii), said suspension of CsNWs is hence dispersed into said aqueous solvent to reach a final amount of CsNWs comprised between 20 and 50 mg / mL, preferably equal to 35 mg / mL or 50 mg / mL.

[0140] According to the invention, the aqueous solvent used in step (ii) is any of the aqueous solvents known in the art or disclosed in the present specification, in particular is water or a mixture of water and ethanol.

[0141] To facilitate solubilization of the CsNWs in the aqueous solvent, step (ii) is preferably performed by mechanical or magnetic stirring, preferably followed by one or more sonication steps, in particular by ultrasonication.

[0142] According to the invention, the acidifying agent used in step (iii) of any of the above processes can be any of the acidifying agents disclosed in the present specification, in particular is acetic acid or lactic acid. The choice of the acidifying agent also depends on the final pH that is desirable for a particular composition according to the invention. In one preferred aspect, in step (iii) an amount or aliquot of an acidifying agent is added to the suspension so as to reach a final pH in the range between 4 and 6.5, preferably between 5.4 and 5.6 or between 5.8 and 6.1. Merely by way of example, in step (iii) a solution of acetic acid 1.6 M can be added to the suspension until a pH in the range of 5.4 and 5.6 is reached.

[0143] In one aspect, the process for the preparation of a composition according to any of the embodiments disclosed herein further comprises adding, in anyone of steps (i) to (iii), at least one additive selected from any of the variants disclosed in the present specification and in the claims in any of the amounts herein disclosed. In a particularly preferred embodiment, the process for the preparation of a composition according to any of the aspects as defined in the present specification and in the claims further comprises a step of adding, in any one of steps from (i) to (iii), at least one polyphenolic compound according to any of the variants illustrated herein.

[0144] Solely by way of example, a polyphenolic compound selected from tannins, lignin, or derivatives thereof, and / or at least one conductive compound selected from biochar, graphene, reduced graphene oxide and carbon dots, and / or a bioactive molecule also acting as activity indicator, i.e. exhibiting a color change upon photodegradation such as methylene blue, and / or at least one pigment or chromophore such as indigo carmine or rosa bengala can be added to the suspension so as to obtain the desired composition.

[0145] An object of the present invention is also represented by a composition obtainable or obtained by means of any of the processes herein disclosed.

[0146] Processes for preparation of films or coatings from CsNWs compositions

[0147] Another object of the present invention is represented by a process for preparing a film or coating comprising deacetylated chitin nanowhiskers (CsNWs), said process comprising the following steps:

[0148] (i) applying a composition according to any one of embodiments disclosed in the present specification onto a target surface or substrate; and

[0149] (ii) allowing the aqueous solvent of said composition to evaporate from the composition so as to form said film or coating. Step (i) of a process for preparing a film or coating according to the present invention can be carried out by means of any of the procedures or technologies known to a person skilled in the art.

[0150] In one preferred aspect, the application of any of the compositions of the invention onto the selected surface or substrate is carried out by a technique selected from solvent-casting, brushing, spraying, painting and / or rolling, or even combinations thereof.

[0151] To impart the resulting film or coating with a predefined size and / or shape, in one aspect, step (i) of the process for preparing a film or coating is performed by depositing a composition according to any of the embodiments disclosed herein into or onto one or more molds.

[0152] Any type of molds known in the art can be used to such purpose, for example molds having rectangular or circular shape, e.g., silicon molds.

[0153] Depending on the type of technique selected for the application of the composition onto the target surface, as well as on the specific application, a person skilled in the art will be able to determine the suitable amounts of compositions to be used in order to obtain a resulting film or coating possessing the desired physical-chemical properties, such as a desired thickness or size.

[0154] Step (i) of any of the above processes is preferably carried out by applying onto the target surface or substrate an amount of said composition so as to form a film or coating that, after evaporation of said aqueous solvent, has a thickness comprised between 5 and 120 pm, preferably a coating having a thickness comprised between 5 and 20 pm or a film having a thickness comprised between 30 and 80 pm, more preferably equal to about 50 pm.

[0155] In one particular aspect, step (i) of any of the above processes is carried out by depositing into or onto a mold an amount of said composition so as to form a film or coating that, after evaporation of said aqueous solvent, has a thickness comprised between 50 and 120 pm, preferably between 30 and 80 pm, more preferably equal to about 50 pm.

[0156] The application of the compositions according to any of the embodiments disclosed herein onto the target surface can also be carried out repeatedly until a desired thickness of the resulting film or coating is obtained, for example can be repeated two or more times so as to obtain the desired thickness. The specific amounts of the components of the compositions (in particular of CsNWs) deposited per area of the target surface can be calculated based on the amount of the compositions used and knowing their density on the target surface.

[0157] In one aspect, evaporation of the aqueous solvent present in the composition can be promoted by carrying out step (ii) at room temperature and atmospheric pressure. Such environmental conditions are particularly advantageous to avoid formation of any undesirable defects in the resulting film or coating, such as bubbles or cracks.

[0158] In one aspect, the application and consequently the spatial distribution of the composition onto a target surface or substrate can be digitally controlled.

[0159] In one aspect, the application of the composition according to any of the variants disclosed herein is carried out in such a way that a homogeneous film or coating of said composition is formed on the target substrate, preferably a micrometer-thin homogeneous film or coating. A person skilled in the art will be able to select the most suitable composition among those disclosed in the present specification, as well as a proper depositing technique and settings in order to obtain a film or coating possessing the desired thickness and physical-chemical properties. In particular, the processes according to any of the embodiments disclosed in the present specification and in the claims allow for the preparation of a film that is free-standing or self-standing.

[0160] In other words, the processes according to any of the embodiments disclosed in the present specification and in the claims allow for the preparation of a film that lacks (i.e. does not contain) any supporting matrix or any supporting compound(s) (such as gels, polymers or polymeric matrix or fillers) other than the deacetylated chitin nanowhiskers, namely a film or coating comprising the sole deacetylated chitin nanowhiskers as supporting elements capable of imparting the resulting film or coating with the desired mechanical-physical properties, thereby making it possible to handle the film as an independent sheet without the assistance of an additional substrate or support.

[0161] Indeed, the processes of the invention particularly involve the use of a composition that comprises deacetylated chitin nanowhiskers as the sole elements capable of imparting the desired strength and mechanical resistance to the resulting film, while lacking the presence of any other compound(s) or precursor(s) capable of forming a supporting matrix or structure, such as gels (e.g. alginate), polymers or polymeric matrix, or fillers.

[0162] According to another aspect, the process for preparing a film or coating according to any of the variant disclosed herein further comprises a step, following step (ii), wherein the formed film or coating is separated from the target surface or substrate. Separation is performed carefully, i.e. , in such a way not to alter nor damage the resulting film or coating.

[0163] Non limiting examples of surfaces or substrates that can be used in any of the processes of the present invention include glass, such as glass slides, as well as metal, cardboard, paper, ceramic, cloth, leather, fiber, fabrics / textiles, wood, plastics, bioplastic and polymeric materials or combinations thereof. Suitable polymeric materials may be any substrate known in the art and may include, for example, polyacrylates, polymethylacrylates, polycarbonates, polystyrenes, polysulphones, polyhydroxy acids, polyanhydrides, polyorthoesters, polypropylenes, polyphosphazenes, polyphosphates, polyesters, cyclic polyolefin copolymers, derivatives thereof or mixtures thereof, which may be surface modified (for example plasma treated) or untreated.

[0164] In one embodiment, the selected surface or substrate is any substrate known in the art that might be used for packaging, in particular packaging of drugs, food and / or beverages.

[0165] In one embodiment, the selected substrate is particularly a substrate or support suitable for biomedical or cosmetic applications, such as an adsorbent material, bandages, gauzes, bands, protection films, patches, fabric, or tissue.

[0166] In one embodiment, the selected surface or substrate is paper or cardboard.

[0167] In another embodiment, the selected surface or substrate is represented by fingernails.

[0168] In another embodiment, the selected substrate comprises or consists of a plant material, such as leaves, stems, branches, fruits, seeds, or roots.

[0169] It forms part of the present invention also a film or coating obtainable by means of a process according to any of the embodiments disclosed in the present specification and in the claims. Preferably, a film obtainable by means of any of the processes disclosed herein is a free-standing or self-standing film. In particular, a film obtainable by a processes according to any of the embodiments disclosed in the present specification and in the claims, lacks (i.e. does not contain) any supporting matrix or any supporting compound(s) other than the deacetylated chitin nanowhiskers, namely comprises deacetylated chitin nanowhiskers as the sole supporting elements capable of imparting such a film with the desired mechanical-physical properties, thereby making it possible to handle the film as an independent sheet without the assistance of a substrate or support. As will be further explained in the following paragraphs, the films or coatings according to any of the embodiments disclosed herein, either as formed onto said surface or substrate or else after being separated from it, can be further processed according to any of the procedures known to a person skilled in the art to be attached or incorporated into any functional material or article such as, for example a biomedical, sanitary, or cosmetic article, or into a suitable material useful for packaging of drugs, food and / or beverages or else for sensing applications.

[0170] Biomedical or cosmetic applications

[0171] As already mentioned in the summary, the authors of the invention have surprisingly found that the compositions according to the present invention, particularly those comprising at least one polyphenolic compound according to any of the variants disclosed herein, can exhibit good biocompatibility and hemocompatibility, as well as advantageous antithrombotic properties, which can be easily modulated by varying the quantity and type of polyphenol used.

[0172] Hence, one aspect of the present invention is referred to a composition according to any of the embodiments disclosed in the present specification and in the claims for use as a medicament.

[0173] In one aspect, the compositions can be used in the treatment or prevention of a skin condition or disease, in particular skin wounds, lesions, abrasions, and / or burns. Given the hemo- and biocompatibility of the compositions of the invention, their application on the skin can indeed be carried out directly onto open wounds, ulcers, and mucous membranes.

[0174] The application of the compositions of the invention on skin is simple and painless and not only protects the skin thanks to a good skin adherence, but in the same way constitutes an active barrier against bacteria, making disinfection unnecessary.

[0175] Preferably, the composition for the use as a medicament according to the above embodiments comprise at least one polyphenolic compound according to any of the variants disclosed in the present specification.

[0176] According to another aspect of the invention, there is herein provided also a biomedical, pharmaceutical, sanitary, or cosmetic article comprising or consisting in a composition, film, or coating according to any of the embodiments disclosed in the present specification and in the claims.

[0177] One preferred embodiment particularly refers to a biomedical, pharmaceutical, sanitary, or cosmetic article in the form of a nail polish comprising a composition according to any of the embodiments disclosed in the present specification, wherein the amount of CsNWs in the composition is preferably equal to at least 50 mg / mL. In one preferred embodiment, said biomedical, pharmaceutical, sanitary, or cosmetic article in the form of a nail polish comprising a composition according to any of the formulations m-1), m-2) or m-3) reported in Tables 26-28.

[0178] As clearly shown in the experimental section of the present specification, such specific compositions once dry on the nail, do not undergo any washout, allowing the user not to avoid or reduce daily hygiene.

[0179] Notably, a composition or article in the form of a nail polish according to any of the embodiments disclosed herein can be used either as a medicament or as a cosmetic product for promoting reinforcement of the nails or for treatment or prevention of onychomycosis and / or onychophagies. To this end, the compositions according to the invention, and consequently the resulting film or coatings, can further include any suitable bittering additive known in the art (e.g., denatonium benzoate), as well as vitamins (e.g., vitamin H, provitamin B5) or any natural additive known in the art to promote nail growth and reinforcement.

[0180] The present invention hence also provides a composition according to any of the embodiments discloses herein, preferably in the form of a nail polish, for use in the treatment of traumatized, weakened, scratched and / or brittle nails.

[0181] Another embodiment refers to a biomedical, pharmaceutical, sanitary, or cosmetic article in the form of a spray comprising a composition according to any of the embodiments disclosed in the present specification. In one aspect, said spray can be directly applied onto the skin of a subject in need thereof, for example for treatment of a skin condition such as those exemplified in the present application and in the claims.

[0182] In one preferred embodiment, said biomedical, pharmaceutical, sanitary, or cosmetic article in the form of a spray comprises a composition according to any of the formulations a-1), a-2), b-1), b-2), c-1), c-2), d-1), d-2), d-3), e-1), f-1), f-2), f-3), g), h) or i) reported in Tables 1-22.

[0183] Another embodiment of the present invention refers to a biomedical, pharmaceutical, or sanitary article in the form of a spray comprising a composition according to any of the embodiments disclosed in the present specification and containing at least one bioactive molecule capable of also behaving as activity indicator such as methylene blue. Such spray formulation can be particularly applied onto a fabric or tissue, for example a laboratory coat, so as to impart it with desirable antimicrobial properties. Advantageously, the presence of an additional antimicrobial molecule, also acting as activity indicator , i.e. capable of exhibiting a color change upon degradation, such as MB, allows to visibly detect the need for discarding or replacing the used fabric or tissue under the laboratory or clinical practice.

[0184] Another embodiment refers to a biomedical, pharmaceutical, sanitary, or cosmetic article in the form of bandages, gauzes, bands, protection films, patches, face mask each one as mono- or multi-layer, which article comprises or consist of a film or coating obtainable according to any of the embodiments disclosed in the present specification.

[0185] Preferably, said film or coating is attached, deposited, or adsorbed onto a substrate or support, preferably a polymeric support, a fabric, a stent, and / or an absorbent material.

[0186] In addition to the compositions, also the films, coatings, or biomedical, pharmaceutical, sanitary articles according to any of the embodiments disclosed herein can be used as a medicament, preferably for the treatment of conditions of the skin of a subject in need thereof, such as for the treatment of wounds, lesions burns and abrasions. To this end, merely by way of example, a composition or article according to the present invention, e.g., in the form of a spray or patch, can be applied topically on the surface of the skin of a subject in need thereof. The application of a composition according to the invention directly onto the skin surface of an individual may advantageously be carried out according to any of the processes disclosed in the present specification to result in the formation of a film or coating on the skin according to any of the embodiments disclosed herein.

[0187] As already mentioned, according to another embodiment, the compositions, films, coatings of the invention can be used for the functionalization or modification of biomedical device or stent. In particular, a film or coating can be formed on the surface of a biomedical device by means of any of the processes disclosed in the present specification, so as to obtain a multifunctional film (or surface coating) aimed at preventing both thrombosis and biofouling phenomena following the implantation of the medical device or during the use of the same at an extracorporeal level.

[0188] In one preferred embodiment, said biomedical device or stent in the form of a film or coating comprises a composition according to any of the formulations b-1), c-1), d-1), d-2), d-3), e-1), f-1), f- 2), f-3), g), h) or i) reported in Tables 3-22.

[0189] Non limiting examples of biomedical devices that can be modified or coated with a composition according to the present invention include a pacemaker, a cardiopulmonary bypass and extracorporeal oxygenation membrane.

[0190] Applications for packaging

[0191] In one aspect of the present invention, the compositions according to any of the embodiments disclosed herein may be used for the fabrication of films or coatings suitable for packaging applications, in particular edible films, or coatings.

[0192] The invention hence provides for a process for the fabrication of a film or coating onto a substrate for packaging of drugs, food and / or beverages, for example onto conventional polymeric packaging materials or biobased / biodegradable packaging materials, wherein said fabrication is carried out by means of a process according to any of the embodiments disclosed in the present specification.

[0193] According to a different embodiment, the free-standing films obtainable by means of any of the processes disclosed in the present specification can be directly used as packaging materials, in particular edible packaging materials. Sensing applications

[0194] In another aspect, the invention provides for a process for the fabrication of a sensor or biosensor comprising at least one step of preparing a film or coating onto a surface or substrate suitable for sensing applications, wherein said preparation is carried out by means of a process according to any of the embodiments disclosed in the present specification.

[0195] For such application the use of compositions comprising a suitable conductive additive according to any of the variants disclosed in the present specification is preferred.

[0196] Applications for preservation of paper

[0197] It forms part of the present invention also the use of a composition according to any of the embodiments disclosed herein for preserving paper, or else for preventing or minimizing degradation of paper, particularly of ancient manuscripts, documents, and photographs. Another object of the invention is hence represented by a method for preventing or minimizing degradation of paper comprising forming onto said paper or onto a paper-based substrate a film or coating by means of a process according to any of the embodiments disclosed in the present specification.

[0198] Examples are reported below which have the purpose of better illustrating the methodologies disclosed in the present description, such examples are in no way to be considered as a limitation of the previous description and the subsequent claims.

[0199] EXAMPLES

[0200] Example 1 - SYNTHESIS OF CHITIN NANOWHISKERS (CtNWs)

[0201] Purified chitin from shrimp shells was used as starting material for the preparation of Chitin nanowhiskers (CtNWs). Otherwise, chitin can be obtained directly from the shells after a purification step that include a basic treatment and a bleaching step to remove the residual proteins and inorganic impurities.

[0202] CtNWs were prepared by hydrolysis of the chitin flakes with 3N HCI under reflux and mechanical stirring for 4 hours. A chitin / HCI solution ratio of 30 g / mL was used. After the reaction, the mixture was cooled to room temperature, the solid was recovered by centrifugation (4700 ref, 15 min) and washed three times with deionized water (in between each washing cycle the solid was recovered by centrifugation as described above). The recovered precipitate was re-suspended in ca. 800 mL of water (based on a starting chitin biomass of 20 g) and divided in smaller aliquots that were sonicated at 40% amplitude for 10 min using a Branson Digital Sonifier (Model 450L, Ultrasonic Corporation) equipped with a 20 KHz Branson probe ending in a horn tip (max amplitude 160W) to obtain an opalescent colloidal suspension. After the sonication step the suspension was diluted with ca. 1 L of water (based on a starting chitin biomass of 20 g) and 1 N NaOH solution was added until a complete precipitation of the CtNWs was achieved. Finally, CtNWs In the form of never-dried gel were subjected to washing / centrifugation cycles until neutrality was reached. Avoiding this last step would result in a dramatical decrease of the physic-mechanical characteristics of the formed films due to the presence of residual salts (usually in the literature this step is carried out by dialysis). At each cycle of washing the speed of centrifugation was progressively increased from 4700 to 20000 ref to obtain a better precipitation of the CtNWs and a more compact gel (this to contrast the pH decrease). The amount of CtNWs in the gel was determined gravimetrically.

[0203] Example 2 - SYNTHESIS OF SUPERFICIALLY DEACETYLATED CHITIN NANOWHISKERS (CsNWs)

[0204] Chitosan nanowhiskers (CsNWs) were obtained by surface deacetylation of CtNWs gel with 12.5 N NaOH under reflux and mechanical stirring for 12 h using a CtNWs / NaOH solution ratio of 60 g / mL. It was noticed that longer reaction times would result in a final dark grey / brown product suggesting a degradation of the polysaccharidic chains. The CsNWs gel was then obtained by washing / centrifugation cycles (as described above for CtNWs), after cooling to room temperature the suspension, until neutral pH. The amount of CsNWs in the gel was determined gravimetrically.

[0205] Example 3 - PREPARATION OF THE FORMULATIONS AND FILM FORMATION

[0206] Two different strategies can be applied to never-dried gel of CtNWs or CsNWs prepared in Example 1 or 2, both exploiting the filming ability of these materials. The first approach is focused on the preparation of free-standing films, while the second on the development of a coatings to be applied on specific surfaces. In both cases the starting point is an aqueous colloidal suspension of nanowhiskers containing various additives to reach the final formulation.

[0207] Free-standing films of CtNWs or CsNWs can be obtained by solvent-casting. Figure 1 shows the different steps to be taken to prepare films from the never-dried gel of nanowhiskers.

[0208] The concentration of nanowhiskers in the formulation plays an important role in the final properties of the films: a too low concentration results in high fragile films, while too high concentrations give very thick, rigid, and inflexible films. A total amount of nanowhiskers equal to 125 mg was found to be optimal for 4.5 cm diameter moulds allowing to achieve a thickness of ca. 50 pm. The gel is first dispersed in water to make a 35 mg / ml suspension and then an acetic acidic solution (1.6 M) is added until a final pH in the range 5.4 - 5.6 is reached. For the film preparation, the concentration of water and nanowhiskers in the gel must be taken into account, i.e., 1.082 g of CtNWs (11.55% w / w) are needed to reach a final amount of 125 mg of nanowhiskers and the remaining 0.96 g are water. Different acids (e.g., hydrochloric acid, lactic acid) or solvents (e.g., ethanol / water mixtures) can be used (Table 29).

[0209] Table 29 - Formulations tested for the preparation of CsNWs- and CtNWS-based films and coatings.

[0210] To facilitate the solubilization, the mixture could be stirred with a magnetic bar and treated in an ultrasonic bath for 15 min at 40°C (Soltec Sonica® ultrasonic cleaner). The suspension was then poured into a circular silicon mould (diameter 4.5 cm, height 2 cm) and stored at room temperature until dryness.

[0211] The formulation of the coatings involves the same steps described above to the obtainment of the colloidal suspension (Figure 1). Ideally, the coating can be applied by brush, spray or roller. In this work the application by brush has been tested. In this case, a denser suspension (in the range 25 - 50 mg / mL) is needed. According to the targeted application of the film and the support on which it is applied, different acids, additives and / or plasticizers can be used (Table 29). Once prepared, the suspension is stable and can be easily stored at room temperature for a period longer than 10 weeks without the formation of any precipitate.

[0212] Example 4 - CHARACTERIZATION OF NANOWHISKERS

[0213] Fourier-transform infrared spectroscopy (FT-IR)

[0214] Fourier transformed infrared spectra were recorded on potassium bromide (KBr) discs, in a spectral range of 4000-400 cm-1using a Perkin Elmer Spectrum One spectrophotometer. Before the analysis, the samples were dried in the oven at 100°C for 4 hours.

[0215] Elemental analysis (CHNS)

[0216] The chemical composition of the starting chitin and of the produced CtNWs and CsNWs was evaluated by CHNS analysis. An elemental Elemental UNICUBE® instrument was used for the measurements. The elemental analysis can be also used for the determination of the degree of deacetylation (XD) of chitin and chitosan deacetylation by the following formula:

[0217] XD= 100*(4-0.583093*WC / N) where: total mass of carbon in sample

[0218] Wc / N = - — total mass of nitrogen in sample

[0219] Thermogravimetric analysis (TGA)

[0220] TGA analysis was performed with a TGA 4000 Perkin Elmer instrument under inert atmosphere (nitrogen) with a flux of 20 mL / min and a heating rate of 10°C / min in the range 50-800°C.

[0221] Dynamic light scattering (DLS) and Zeta Potential

[0222] DLS and zeta potential analysis were performed with a Malvern Zetasizer Ultra to evaluate the size and surface charge on the nanowhiskers. The sample was suspended with a concentration of 1 mg / mL at pH 4.02.

[0223] Scanning electron microscopy (SEM)

[0224] SEM images of the nanowhiskers and films were collected on unsputtered samples by Field emission scanning electron microscopy (FE-SEM) with a Zeiss Sigma. 1 kV and an InLens detector were used for the analysis.

[0225] Atomic force microscopy (AFM)

[0226] The analysis was carried out with Park XE-7 (Park Systems Inc.) coupled with SmartScan software. The preparative phase consists in the deposition of a drop (10 pL) on a mica support and then, after 15 seconds, in the removing of unabsorbed material by immersing the sample in water for additional 15 seconds. Before the analysis the samples were dried at room temperature.

[0227] Example 5 - CHARACTERIZATION OF FILMS

[0228] Film thickness

[0229] Film thickness was determined with a vertical micrometer (sensitivity ± 10 pm). Contact angle

[0230] The contact angle measurements were carried out on a First Ten Angstroms (FTA) 1000 Drop Shape instrument. Static mode was used to determine the degree of hydrophobicity after 3 s from drop deposition. The water absorption kinetics were evaluated by measuring the static contact angle over a time frame of 2.5— 5 minutes.

[0231] Water absorption

[0232] The amount of water absorbed by the films was calculated gravimetrically. The film, previously dried in a static oven at 30°C under vacuum till constant weight (about 2 h are needed), were weighed, soaked in deionized water, and placed in an ArgoLab Ski4 shaker at room temperature under stirring (150 rpm). At predetermined time intervals (10 min are sufficient to reach equilibrium) the films were withdrawn, wiped with a Kimtech paper, and weighed. The absorbed water was calculated according to the following formula:

[0233] Changes in the film size were measured by a micrometer (length and width) and a vertical micrometer (thickness, see above).

[0234] Tensile tests

[0235] Stress-strain measurements were performed on rectangular films (5 mm wide and 0.05 mm thick) using an MTS Insight moving beam dynamometer machine equipped with a 100 N load cell. The gauge length was equal 20 mm, and the crosshead speed was set to 0.5 mm / s.

[0236] Tensile elastic modulus was determined from the first linear segment of the stress-strain curve. At least five replicates were run for each sample and the results were provided as the average ± standard deviation.

[0237] 31P-NMR

[0238] A Bruker 300 MHz NMR spectrometer was used for the31P NMR analyses. The sample (previously dried and accurately weighed) was phosphitylated with the use of 2-chloro-4, 4,5,5- tetramethyl-1 ,3,2-dioxaphospholane, according to a procedure reported in the literature (Argyropoulos D.S. et al., Quantitative31P NMR Analysis of Lignins and Tannins. J. Vis. Exp. 2021, (174)). A mixture of deuterated chloroform and anhydrous pyridine in volume ratio 1 :1.6 was used as solvent and cholesterol was used as internal standard.

[0239] Release of polyphenols in aqueous media

[0240] The CsNWs films loaded with the different polyphenols (Table 29) were soaked in Milli-Q water and placed in an ArgoLab Ski4 shaker at 37°C under stirring (150 rpm). Samples were withdrawn at predetermined time intervals over a time span of 5 days and analyzed using a LIV-VIS spectrophotometer (Shimadzu UV-1800) to evaluate the amount of polyphenol released in the medium. For each polyphenol a calibration curve was constructed in the concentration range 0.0025 - 0.02 mg / mL.

[0241] Biocompatibility studies

[0242] Indirect contact test

[0243] To evaluate the cytotoxicity ascribable to the release of purified hardwood lignosulfonate, MIHLS films and their control counterpart (CsNWs and CsNWs-G) were cut in square-shaped pieces (1cm2area), placed in 24-well plates, and sterilized for 60 min per side under UV light to prevent contamination. After quick wash with 1 * phosphate buffer solution (PBS) (137 mM NaCI, 2.7 mM KCI, 4.3 mM Na2HPC>4, 1.4 mM Nab^PC , pH 7.4), each type of films was incubated with 1 mL of cell culture media at 37°C, 5% CO2. After 24 h of incubation, culture media (the extracts) was completely collected. NIH-3T3 were previously seeded at a density of 1 x 104cells per well in 96- well plates and incubated without (control) or with increasing serial dilutions of the extracts collected after 24 h from the films. After 24 hours (h) of incubation, cytotoxicity was assessed by the quantitative 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test (Sigma-Aldrich) according to manufacture”s instructions. In brief, the culture medium was replaced by 100 pL Dulbecco's Modified Eagle Medium (DMEM) high glucose supplemented with 10 pL of a 5 mg mL-1solution of MTT in 1 xPBS, and the cell cultures were incubated for 3 h. Viable cells can reduce MTT into formazan crystals. After the addition of 100 pL of solution C (2-propanol and HCI 0.04 %), the well plate containing the cultured cells was incubated at 37°C for 30 min. Aliquots of 100 pL were sampled, and the absorbance was measured at 595 nm with a reference wavelength of 650 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA). A standard curve of cell viability was used to convert the results expressed as percentage related to the control set equal to 100 %.

[0244] Direct contact test

[0245] Prior to cell seeding, all films were shaped into suitably sized pieces, sterilized for 60 min per side under UV light, fixed with CellCrown support for 24-well plates (Scaffdex, Tampere, Finland) to prevent them floating, and then incubated with culture medium at 37°C for 24 h before cell seeding. Afterwards, a drop of cell suspension containing 1 x 105cells was added onto the top of each film, and after 30 min, 1 mL of culture medium was added to cover the films. NIH-3T3 cultured on tissue culture plates (TCPS, Tissue Culture Polystyrene Plate) were used as positive controls. Resazurin- based assay (TOX8-1 KT, Sigma-Aldrich) was performed to assess cell viability on the different film at 24 h from cell seeding. According to manufacturer instructions, resazurin solution was added in 1 :10 ratio with respect to culture volume to each well plate and incubated for 4 h at 37 °C in 5% CO2. At the end of incubation time, absorbance of the samples was measured at a wavelength of 600 nm with a reference wavelength of 690 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The cell viability assay was performed in parallel both on unseeded-scaffolds and on seeded-TCPS. Cell viability and morphology after 24 h was also assessed by optical light microscopy by acquiring different fields for each sample and by immunofluorescence. Concerning immunofluorescence protocol, cell-seeded scaffolds were washed with PBS, fixed with 4% (w / v) paraformaldehyde solution (PFA) for 30 min at 4°C, and then permeabilized with 0.1 % Triton X-100 for 5 min. In order to detect focal adhesion, cells were incubated with primary mouse anti-p 1 integrin (1 : 100 in 1 % BSA, NSJ Bioreagents, San Diego, CA). Afterwards, samples were incubated with specific secondary antibody for immunofluorescence at a concentration of 1 : 500 in 1% BSA. Hoechst 33342 (2 pg mL-1) was used for nuclei staining. The images were taken using a TCS SP8 confocal microscope (Leica Microsystems, Bensheim, Germany) equipped with a digital image capture system at 40* magnification.

[0246] Hemocompatibility of the film Clotting time

[0247] Prothrombin time (PT) and activated partial thromboplastin time (APTT) were determined by measurement of clotting times of platelet poor plasma (PPP) after incubation with the different films according literature protocols [Horakova J. et al., Comprehensive assessment of electrospun scaffolds hemocompatibility. Mater. Sci. Eng. 2018, 82 330-335, Van W. et al., Comparison of Coagulation Activity Tests In Vitro for Selected Biomaterials. Artif Organs. 2002, 26(6), 506-11]. Sterilized tested materials were cut into squares of 1 x 1 cm size and placed in a test tube followed by addition of 300 pl PPP. As controls, 300 pl of PPP was incubated in test tubes without any material present. After 60 min of incubation in 37 °C, the film was removed from test tube and both PT and APTT were measured using automatic analyzer BCS 5100XP (Siemens) according to the manufacturer's instructions.

[0248] Human fibrinogen absorption

[0249] Enzyme-linked immunosorbent assay (ELISA) was performed to measure the human fibrinogen (hFg) absorption on each type of film. Sterilized films were shaped into suitably sized pieces (0.32 cm2) and inserted in a 96-microtiter well. Afterwards, each film was incubated with a 100 pL solution of hFg (10 pg / mL), overnight at 4 °C. After three washes with PBS containing 0.1 % (v / v) Tween 20, the samples were blocked by incubating with 200 pL of PBS containing 2% (w / v) BSA for 2 h at 22 °C. The samples were subsequently incubated for 1 h at 22 °C with 100 pL with horseradish peroxidase (HRP)-conjugated goat antibody anti-hFg (1 :10000 dilution in 1% BSA, ROCKLAND). Reaction development with 200 pL / well HRP chromogenic substrate (TMB; Sigma- Aldrich, St. Louis, MO, USA). Absorbance at 450 nm was measured with CLARIOstar ® Plus Multimode Microplate Reader (BMG Labtech, Ortenberg, Germany). HFg absorbed on high binding 96- well microtiter was used as positive control whereas a negative control was prepared with well bottom bovine serum albumin (BSA) coating. The optical densities from each sample were plotted against a calibration curve generating with increasing concentrations of purified human fibrinogen protein (from 500 ng to 10 pg / mL).

[0250] Platelet adhesion Quantification of platelets adhered to films was determined using an in vitro using a lactate dehydrogenase assay (LDH, Sigma Aldrich). This method is based on the reduction of NAD by the action of LDH, which is a stable cytoplasmic enzyme present in cells. It is rapidly released into the lysing solution upon damage of the plasma membrane. The use of a toxicology assay kit allows quantification of the number of platelets as a function of the amount of LDH released. Human platelets (500 pL of 2 x 107platelets / mL in Tyrode’s buffer) were incubated on the films, previously inserted in sterile a 24-wells, for 1 h at 37°C. Platelets seeded on BSA (1%), and Collagen-I (10 pL / mL) coated-well were used as negative and positive controls, respectively. The platelet suspension was then removed from the surfaces and test surfaces were washed three times with PBS 1 x. Films were moved to fresh wells and the LDH release was quantified after lysis of the adhered platelets using 1.5% Triton X-100 in PBS according to the manufacturer’s instructions. The absorbance was measured at 450 nm against using a microplate reader. A standard curve, generated in parallel using known platelet concentrations, was used to express the number of platelets adhered per cm2. Immunofluorescence studies was performed to visualize platelets adhesion on the different surfaces. Briefly, PFA-fixed platelets were permeabilized using Triton-X 100 (0.025% in PBS), three time washed and incubated with phalloidin-TRITC (1 :500 in PBS, Sigma Aldrich). The samples were then observed by a confocal laser scanning microscope (TCS SP8, Leica Microsystems, Bensheim, Germany, Oil 63x).

[0251] RESULTS AND DISCUSSION

[0252] As described in the above examples, the preparation of the CtNWs involves an acid hydrolysis of the chitin amorphous domains and the isolation of the crystalline part. A subsequent step of surface deacetylation yields the CsNWs. From 20 g of chitin flakes 150 g of never-dried gel were obtained with a concentration of CtNWs equal to 7.3 wt%. The concentration of CtNWs in the gel was determined by gravimetric analysis after drying the sample at 100°C until complete evaporation of the water. The concentration of CtNWs is affected by the speed of centrifugation. As expected, a higher centrifugation speed allows to reach a higher concentration of CtNWs in the gel, up to a value equal to 11.6% for CtNWs and 13.1% for CsNWs with a centrifugation speed of 20000 ref. To verify the success of the treatment, CtNWs, CsNWs and pristine chitin were subjected to elemental analysis (CHNS). The results are reported in Table 30.

[0253] Table 30. CHNS data of chitin, CtNWs and CsNWs samples.

[0254] The results demonstrate that the hydrolysis process of the amorphous part of the polymer did not lead to any variation in the N / C ratio. On the contrary, the subsequent deacetylation step caused an increase of the N / C ratio due to the removal of the -CH3CO group. The data obtained agree with those reported in the literature. The CHNS data also evidence a greater quantity of sulfur in the pristine chitin sample. At a biological level, sulfur is present in the amino acid cysteine and methionine, constituents of various proteins, as well as in active sites of enzymes and proteins. Since the chitin used in this study was extracted from the shrimp exoskeleton, it can be hypothesized that the presence of sulfur is related to protein residues. The decrease in the sulfur content shown in the CtNWs and CsNWs samples could be ascribed to the hydrolytic treatment, in which not only the removal of the amorphous fraction of chitin occurs, but also the depolymerisation of the protein residues into amino acids that are subsequently removed during the washing cycles. Furthermore, CHNS analysis allowed for the determination of the degree of deacetylation in both the CtNWS and CsNWS (see experimental section for details), respectively equal to 4.6% and 36.8%, confirming the success of the deacetylation step.

[0255] To evaluate the morphology and the size of the nanowhiskers, atomic force microscopy (AFM, Figure 2A) and dynamic light scattering (DLA, Figure 2B and Figure 2C) analysis were conducted. The typical rod-like shape of nanowhiskers can be observed from the images collected via AFM. The tendency of the nanowhiskers to aggregate in layered microstructures is also visible (Figure 2A). The DLS analysis did not reveal significant size differences between the two samples, both showing a length of about 320 nm, in agreement with previous studies. To better evaluate the stability of the nanowhiskers in the liquid formulation, zeta potential analysis was performed. CtNWs and CsNWs were dispersed in acidified water (pH 4.02) at a concentration of 1 mg / mL prior to analysis. In both cases a value of +54 mV was recorded, suggesting that a stable colloidal suspension can be prepared, since Z-potential values in the range between ±40 and ±60 mV usually indicate that aggregation and flocculation phenomena are prevented. These data were supported by the observation that suspensions of CsNWs and CtNWs did not show any evident formation of precipitate upon storing at room temperature for a period of 10 weeks.

[0256] FT-IR and TGA analysis were carried out to further characterize the prepared materials in terms chemical structure and thermal stability. All the bands of the different the functional groups of CsNWs and CtNWs were visible on the FT-IR spectra. Specifically, those ascribable to the C=O stretching of the primary amide (1650 cm-1) and to the hydroxyl group stretching (3450 cm-1) are the most characteristic. As to the TGA curves, the first weight loss step, up to about 100°C, is due to the evaporation of residual water. Up to 250°C, no weight loss is then observed, indicating that no degradation occurs in both samples. A second weight drop highlighted in the range 250°C-400°C, can be attributed to the dehydration of the saccharide ring and to the decomposition of the acetylated (in the CtNWs) and deacetylated (in the CsNWs) units. The weight loss observed at temperatures above 400°C is finally linked to the complete decomposition of the polysaccharide residues.

[0257] CsNWs FILMS FOR BIOMEDICAL APPLICATIONS

[0258] CsNWs were used to prepare free standing film via a solvent-casting procedure, involving the addition of different additives to the formulation to modulate the mechanical behavior and provide specific functional characteristics (Table 29). Initially, to optimize the concentration of nanowhiskers in the aqueous solution, films were prepared using the sole CsNWs (no additives were added). Concentrations below 20 and above 50 mg / mL led to the obtainment of very fragile films, susceptible to fracture upon bending. The final optimal amount of CsNWs for the preparation of films of 15.2 cm2was found to be 125 mg (with a concentration of 35 mg / mL). The use of CtNWs led to the formation of highly fragile films as compared to CsNWs. This result can be explained based on the higher intermolecular interactions between the CsNWs whiskers with respect to the CtNWs whiskers because of the removal of the surface acetyl groups. The addition of plasticizing agents such as glycerol, triethyl citrate, etc. can on the one hand increase the flexibility of the matrix, on the other counteract the stiffening effect provided by other additives (such as polyphenols) capable of interacting via weak interactions, e.g., hydrogen bonding, with the nanowhiskers. Specifically, a concentration of glycerol equal to 30 wt% (sample CsNWs-G) with respect to the amount of nanowhiskers was found optimal and allowed the introduction of concentration of other additives as high as 20 wt% without compromising the mechanical properties of the films, otherwise recorded for lower concentrations of glycerol (results not shown). Different molds (in terms of shape and material used) and drying temperatures and pressures were tested to optimize the casting procedure. Circular and smooth silicone molds allowed the production of easily detachable defect-free films (Figure 4).

[0259] As to the temperature and pressure, the best conditions to obtain bubble-free films and to avoid any possible degradation of the additives are slow evaporation at room temperature and atmospheric pressure. On the other hand, the use of a ventilated oven or the solvent evaporation under vacuum resulted in the formation of defective films (showing bubbles, cracks, etc.).

[0260] Three different water-soluble natural polyphenols were chosen as specific additives due to their well-known properties (i.e., antimicrobial, antioxidant, etc.). Specifically, green tea extract (epigallocatechin gallate), tannic acid and lignosulphonate, respectively for monomeric tannin, hydrolysable tannin and lignin were selected, and used for the preparation of different films, respectively named EPCG, TAN and MIHLS (see Table 29). Furthermore, the use of monomeric, oligomeric and polymeric polyphenols allowed to evaluate the effect of the different molecular weight on the functional properties of the films. Figure 5 shows the images of films produced under optimized conditions and the SEM micrographs of the film surface, where the single nanowhiskers are clearly visible.

[0261] The films were characterized in terms of surface wettability and water absorption by water contact angle and gravimetric measurements. The results obtained are shown in Figure 6.

[0262] The introduction of polyphenols in the formulation led to a slight decrease of the contact angle from 88° for CsNWs-G to 80°, measured in the case of the MIHLS20 sample. The increased surface wettability is due to the presence of the hydroxyl groups contained in the polyphenols. It can be observed that the higher hydrophilicity is directly correlated to either the concentration of the OH groups of the polyphenol, as measured by31P-NMR (Table 31), or to the amount of polyphenolic additive in the formulation: the higher the concentration of OH groups, the lower the contact angle, and the higher the amount of polyphenol, the lower the contact angle. In this last case, an important role is also played by the sulphonate groups present in the MIHLS. Furthermore, over the time frame of 5 mins, a decrease of the volume of the water drop deposited on the surface of the film is visible for all samples (Figure 6B). This phenomenon, due to the water absorption within the film, depends on the formulation. Specifically, CsNWs-G showed the higher decrease, while the polyphenolcontaining samples a lower decrease, which can be correlated with the concentration and extension of hydrogen bonds that the polyphenol can form with the nanowhisker matrix (see below).

[0263] Table 31. Molecular weight and content of hydroxyl and carboxylic groups in the polyphenolic additives, as measured by31P-NMR.

[0264] *number average molecular weight as measured by gel permeation chromatography [Da].

[0265] As to the water adsorption tests, the gravimetric measurements (Figure 6C) show a weight increment of ca. 35% for the CsNWs-G sample. The introduction of polyphenols leads to a much lower enhancement of the weight as variations in the range 1-17% have been recorded. It is interesting to note that the water absorption process reaches the equilibrium already after 10 min, while a slight weight decrease over the course of 24 hours can be observed as due to the diffusion of glycerol and polyphenols from the films to the aqueous medium (results not shown).

[0266] Therefore, after 10 min of incubation in water, length, width, and thickness of the films were measured and compared to the initial values (before incubation). As it can be seen from the data reported in Figures 6D-E, the film thickness significantly varies (Figure 6D) while no appreciable variations in length and width are observed.

[0267] A lower increment of thickness can be observed for the polyphenol-containing films with respect to the CsNWs-G sample, in agreement with the gravimetric measurements. More in detail, as to the samples containing 1% of polyphenols, the lower weight and thickness increment has been observed for the TAN1 films. Additionally, by increasing the concentration of MIHLS, a progressively lower variation of weight and thickness can be highlighted.

[0268] Recent studies reported in the literature have demonstrated that chitin nanowhiskers are arranged in stratified lamellae and that this arrangement is an intrinsic property of the nanowhiskers themselves. On these bases, it can be hypothesized that water intercalates between the layers, leading to an increase of the thickness of the film.

[0269] The different thickness variations observed for the films under study can be explained based on the presence of intermolecular hydrogen bonds established between the hydroxyl groups of the polyphenols and the amino groups of the nanowhiskers, which keep the different film layers more closely linked to each other, thus hampering the swelling effect due to the intercalation of water molecules (Figure 7).

[0270] This behavior is correlated to the chemical structure of the polyphenol additive introduced in the film and, specifically, to the concentration of hydroxyl groups and to the molecular weight of the additive (Table 31). The higher the concentration of OH functionalities, the higher the amount of hydrogen bonds that can be formed. Furthermore, the higher the molecular weight, the higher the extent of interactions and the number of nanowhiskers that can be involved. On these bases, the better results are achieved by TAN, which displays the best combination of these two characteristics among the used polyphenols.

[0271] The tensile tests of the prepared films evidenced a clear dependence of the mechanical behavior on the characteristics of the polyphenol present in the formulation (Figure 8 and Table 4) in agreement with the water adsorption analysis.

[0272] Specifically, the presence of polyphenolic moiety in the film generates an enhancement of the elastic modulus (E) of the films. The higher increase has been recorded for the TAN1 sample, while comparable results can be observed for the EPCG1 and MIHLS1 (Table 32). The stress at break (Ob) of the TANI sample is higher than that of CsNWs-G, EPCG1 and MIHLSI samples, while in terms of strain at break (£b) a slight decrease has been observed for all the films containing polyphenols with respect to the CsNWs-G. This behavior well correlates with the concentration and extent of hydrogen bonds, as above described, which act as crosslinking points raising the film stiffness.

[0273] A slight increase of the stress at break can be highlighted by increasing the concentration of MIHLS in the film formulation, confirming the role of the hydrogen bonds, while no significant differences in terms of elastic modulus and elongation at break were detected.

[0274] Table 32. Elastic modulus (E), stress at break (Ob) and elongation at break (£b) for CsNWs- based films obtained from tensile tests.

[0275] Subsequently, the release profile of the polyphenolic moieties present in the film was analyzed in aqueous media. Since the targeted application is in biomedicine, initially the films were soaked in buffered media simulating the physiological environment (Phosphate Buffer Saline at pH = 7.4) and the pH of the skin (Sodium acetate buffer at pH = 5.0). Unfortunately, the high metal-ion complexing ability of the polyphenols led to the quick formation and subsequent precipitation of complexed species, thus preventing a correct measurement of the concentration of polyphenols in the medium. Therefore, the release studies could only be conducted in pure water (Milli-Q water was used for the experiments). The results obtained are reported in Figure 9.

[0276] For shorter incubation times (up to about 50 min), a comparable release rate can be observed in the case of the different polyphenols (Figure 9A). At higher incubation times, a different release trend could be observed in the case of the film containing EPCG since its concentration in the aqueous medium apparently decreased after reaching a plateau of about 4.5% of release, while for both TAN1 and MIHLS a plateau at a concentration of 3 pg / mL ca. can be observed. To explain this result the instability of EPCG in aqueous media should be taken into consideration, this last following two major degradation pathways epimerization and auto-oxidation. Specifically, in aqueous environments with pH < 5.5, temperature > 50°C and for concentrations higher than 10 mM, the epimerization rate of epigallocatechingallate into gallocatechingallate is significant. On the other hand, he auto-oxidation reaction occurs mainly for pH > 5.5 and consists in the transformation of EPCG into the asinensin A, essentially a dimer of EPCG. This reaction is accompanied by change of the color of its aqueous solution from pale pink to brown [ Hou Z. et al., Mechanism of action of (-)- epigallocatechin-3-gallate: auto-oxidation dependent inactivation of epidermal growth factor receptor and direct effects on growth inhibition in humanesophageal cancer KYSE 150 cells. Cancer Res. 2005, 65 8049-56\. Since the release tests were conducted at room temperature in Milli-Q water and EPCG concentrations lower than 10 mM were reached, it can be inferred that the autooxidation reaction plays a major role in determining the apparent decrease of EPCG in the aqueous solution. It is important to mention that some biological effects have been directly attributed to EPCG degradation products, rather than to EPCG itself [ O. Krupkova et al. Stability of (-)-epigallocatechin gallate and its activity in liquid formulations and delivery systems. J. Nutr. Biochem. 2016, 37 1-12], and that the product of epimerization, gallocatechingallate, has been described as having the same biological properties of the cis molecule.

[0277] It can be noticed that the same auto-oxidative reaction, leading to colour changes, was also observed in the film upon storage in air at room temperature for longer than 30 days. This film was also studied in terms of biocompatibility analysis cytotoxicity and compared to a freshly casted film (see notes in Table 29).

[0278] As, expected, by increasing the concentration of polyphenol in the formulation a higher amount of the additive was released in water, which reached a concentration of 60 pg / mL after 1800 hours of incubation. (Figure 9B). However, in terms of percentage of polyphenols released from the film matrix, these values are in all cases below 10% further confirming the presence of interactions between the nanowhiskers and the polyphenols.

[0279] Various film formulations (Table 29) have been tested to evaluate their biocompatibility, with the lignosulfonate-containing films showing the best properties. Therefore, only the results obtained with these specific films are discussed in detail. In vitro indirect and direct-contact tests were performed with murine fibroblasts cell line NIH3- T3. This cell line is commonly used in cytotoxicity tests and is generally considered as a suitable cell model for studying material biocompatibility [Rejmontova, P. et al., Adhesion, Proliferation and Migration of NIH / 3T3 Cells on Modified Polyaniline Surfaces. Int. J. Mol. Sci. 2016, 17, 1439; Rajaratanam D.D. et al., In vitro cytotoxicity of superheated steam hydrolyzed oligo((R)-3- hydroxybutyrate-co-(R)-3-hydroxyhexanoate) and characteristics of its blend with poly(L-lactic acid) for biomaterial applications. PLoS One. 2018, 26; 13(6)]. The desired biomaterials should be biocompatible and not cause any negative effects on the surrounding tissues. To evaluate the effect of purified hardwood lignosulphonate (MIHLS) released on cell viability, cells were incubated with increasing serial dilutions of the extracts collected by incubating each film for 24 hours with the culture medium (Figure 10A). Interestingly, cell viability was not reduced when cells were treated with film extracts, except for MIHLS20 extract, which caused an approximately 30% reduction in cell viability compared to CsNWs, CsNWs-G and untreated cells (Figure 10B). This evidence is therefore very important because it shows that the risk of exposure to the toxic elements released by the films is absent or very low.

[0280] The direct contact test (Figure 11 A) showed a significant reduction in cell viability on all films tested compared to TOPS (Figure 11B, *p<0.05). Compared to CsNWs and CsNWs-G, lower viability was determined on MIHLS, and mostly on MIHLS20, suggesting that lignosulphonate incorporation affects the cell adhesion capacity of the films (Figure 11B, *p<0.05). Immunofluorescence images obtained by incubating adherent cells with nuclei staining and with an antibody specific for integrin pi (the main transmembrane binding protein of focal adhesion, in green) [Hyvari L. et al., Focal Adhesion Kinase and ROCK Signaling Are Switch-Like Regulators of Human Adipose Stem Cell Differentiation towards Osteogenic and Adipogenic Lineages. Stem Cells Int. 2018, 13], clearly showed a significant reduction in cell viability and adhesion on the films compared to TCPS. In particular, less adhesion was observed especially on MIHLS films, as demonstrated by the significant reduction in the number of nuclei and fluorescence intensity of integrin pi , suggesting a lower and delayed cell adhesion process on these surfaces compared to TCPS, CsNWs and CsNWs-G (Figure 11 C). Optical images supported the viability and immunofluorescence data: flattened adherent cells homogeneously covering the TOPS and lower adherent cells unevenly distributed both in number and morphology (flattened or rounded) on the films (Figure 12).

[0281] For blood compatibility tests, clotting time measurement (Figure 13), absorption of human fibrinogen (Figure 14) and human platelets adhesion (Figure 15) were performed. The measurement of PT and APTT was conducted to establish the effect of films on coagulation. PT and APTT represent the extrinsic and intrinsic pathways of blood coagulation, respectively, and are measured to assess the blood's ability to form clots. A prolonged or reduced time of PT or APTT indicates greater or lesser anticoagulant activity of the materials tested. All films tested did not affect PT (Figure 13A) and APTT (Figure 13B): PT and APTT values remained within the normal clinical reference range (reference values of PT and APTT, 10-12 and 20-32 respectively), like the value measured on the control group.

[0282] Fibrinogen was identified as the key protein in the biomaterial-induced platelet adhesion process [Liwei Y. et al., Galloyl groups-regulated fibrinogen conformation: Understanding antiplatelet adhesion on tannic acid coating. Acta Biomater. 2017, 64 187-199]. All films tested showed a lower amount of hFg than the control group (hFg absorbed on the high binding well) (Figure 14, *p<0.05). Of note, MIHLS films support significantly lower fibrinogen adsorption than CsNWs and CsNWs-G (*p<0.05), indicating that the chemistry of the films may explain the different fibrinogen binding mechanism observed in this study.

[0283] Regarding the platelet adhesion test, the platelet adhesion studied with the LDH assay was drastically reduced compared to the adhesion of platelets to MIHLS films, CwNWs, CwNWSs-G and the positive control (adherent platelets on collagen-coated well) (Figure 15A). The result is in line with the immunofluorescence, which shows the lower adhesion of platelets on the MIHLS film. (Figure 15B).

[0284] Previous studies have shown that the chitosan-coated surface inhibits cell attachment and that the modification of amine and hydroxyl groups increases its non-adhesive properties [Da-Eun K.et al., Application of Hexanoyl Glycol Chitosan as a Non-cell Adhesive Polymer in Three- Dimensional Cell Culture. ACS Omega. 2022, 7 (22), 18471-18480]. In conclusion, this study demonstrates that the CsNWs-based films do not release toxic compounds, and can reduce protein adsorption and platelet adhesion, thus resulting in biocompatible biomaterials with functional characteristics.

[0285] COATINGS FOR THE CONSERVATION OF MANUSCRIPTS

[0286] The conservation of manuscripts is focused on minimizing the degradation of the paper mainly caused by physical (temperature, light, humidity) and biological agents (insects, bacteria, and molds). In this context, the goal is the formulation of a nanostructured chitin-derived multifunctional coating capable of counteracting the different degradative processes thanks to the numerous properties of the starting material. The formulation, based on CsNWs, was optimized as described in Figure 16.

[0287] Of the different parameter varied during the optimization process, the pH value needed for the dispersion of the NWs has required greater attention to avoid any kind of degradation of the paper substrate catalyzed by acid hydrolysis. Specifically, the use of lactic acid and the insertion of a further sonication step before the coating process has allowed to obtain a colloidal suspension with a pH between 5.8 and 6.1. The use of lactic acid is also justified by its antimicrobial activity as it acts as a permeabilizer of the gram-negative bacterial outer membrane and thus enhancing the effect of other antimicrobial substances [Alakomi H.L et a!., Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Appl Environ Microbiol. 2000, 66(5) :2001-5], The application of the coating involves the use of 0.5 ml of the prepared suspension and the application by a brush in all directions of a 36 cm2paper sheet (1 Coating layer) previously placed on KAPTON (polyamide) film. The use of higher quantities of suspension and different supports led to the adhesion of the paper sheet on the support due to permeation of the coating through the cellulose fibers. After the drying time (approx. 1 h at room temperature in air), the formation of a transparent coating layer on the surface of the paper is visible (Figure 16). Thickness measurements highlighted that a first spreading of the coating does not lead to any thickness increase. Contrarily, the use of additional 0.5 ml of the coating formulation (2 Coating layers) results in an enhancement of the thickness of the paper of about 5 microns on the surface on which it is applied. To evaluate the effectiveness of the deposited coating, two different tests were carried out. The first consists in drawing a flower on the paper sheet before the coating deposition and, after the coating has dried, to test whether the drawing can be erased by means of a rubber. The results show that the coating effectively protects the underneath surface as the drawing can’t be removed (Figure 17A). The second test is carried out to evaluate the permeation of a methylene blue solution through the paper after the coating deposition.

[0288] Figure 17B clearly shows that the dye penetrates the paper only in the areas not covered with the coating, while the coating avoids any methylene blue diffusion through the paper sheet.

[0289] Paper samples after coating deposition were characterized from a physicochemical point of view by measuring surface wettability and water absorption. The deposition of one or more coating layers does not significantly alter the surface wettability of the paper (Figure 18A). With regards to the water absorption, as already demonstrated in the case of the test with methylene blue, the deposition of a single coating layer is already sufficient to prevent the permeation of water through the paper (at least over the tested time frame of 150 s). To evaluate the effect of the deposition of the coating, tensile tests were carried out on coated and uncoated paper sheets along longitudinal and transverse direction.

[0290] Table 33. Elastic modulus (E), stress at break (ob) and elongation at break (eb) for uncoated and coated paper obtained from tensile tests. The deposition of one or two layers of coating does not alter the elastic modulus (E) along the longitudinal direction of the fibers, while it enhances the stiffness of the paper along the transverse direction, as an increase of the E value of about 25%, independently whether one or two coating layers are present, is observed. No significant changes in terms of elongation at break (£b) have been recorded, while a clear reinforcing action is exerted by the CsNWs, as the stress at break (Ob) is almost doubled both along the longitudinal and transverse direction.

[0291] NAIL POLISH FORMULATIONS BASED ON CsNWs

[0292] The film-forming ability of the CsNWs can be also used to produce natural, water-based, and non-toxic nail polishes, which can be employed for beauty purposes, by simply adding pigments / dyes, or as a medical device for the treatment of onychomycosis, due to the well-known antimicrobial properties of chitosan. For these uses, denser CsNWs suspensions (50 mg / mL) should be prepared.

[0293] Different solvents have been used to prepare the CsNWs suspensions. Specifically, CsNWs were dispersed in water (W) and water / ethanol mixtures: water / ethanol = 3 v / v (WE3) and water / ethanol = 2 v / v (WE2). A simple experiment has been carried out by applying the nail polish on all the five fingers of a hand and testing its removability every 10 mins (after a first drying period identified in 10 mins for water / ethanol mixtures and 20 min for water, i.e. , a time frame to ensure polish resistance to gentle wiping with dry absorbent paper). The test is carried out on one fingernail at a time by washing it under running water and, subsequently, if the treatment is not sufficient, by wiping with wet absorbent paper (Figure 19).

[0294] If water is used, 60 min (including the first drying period) are sufficient to achieve complete dryness of the polish. Indeed, after 30 min the polish can still be easily washed off by running water, while after 40 and 50 min the polish is only partially removed by the above-mentioned procedure. As to the water / ethanol mixtures, a shortening of the first dying period (halved from 20 to 10 min) and a simpler applicability (due to the lowering of the surface tension of the solvent) can be observed. 60 min are needed to reach complete dryness in the case of the WE3 sample, highlighting no significant differences with the W sample, while an increase of the content of ethanol in the formulation (WE2 sample) results in a worsening of the resistance of the polish, since it can be removed by simple washing under running water even after 60 min from the application.

[0295] Once fully dry, the polish is homogeneously distributed on the nail, no defects are visible and it shows an excellent adhesion to the substrate since it nicely withstands normal daily operations (washing with water, repeated use of soaps and detergents, impacts with surfaces) without any appreciable worsening of the visual appearance. Acetone is not suitable for the polish removal.

[0296] Figure 20 demonstrates the visual appearance of nails treated with the product (both without and with the addition of organic dyes). The application of three coats ensures perfect results in terms of homogeneity and nail covering.

Claims

CLAIMS1. A composition comprising deacetylated chitin nanowhiskers (CsNWs), an acidifying agent, an aqueous solvent and at least one polyphenolic compound, preferably wherein said composition further comprises a plasticizer.

2. The composition according to claim 1 , having a pH of between 4 and 6.5.

3. The composition according to claim 1 or 2, wherein said plasticizer is selected from the group consisting of glycerol, triethyl citrate, polyethylene glycol (PEG), glycerol oligomers, and combinations thereof.

4. The composition according to claim 3, wherein said plasticizer is glycerol.

5. The composition according to any one of claims 1 to 4, wherein said acidifying agent is selected from acetic acid, lactic acid, azelaic acid and hydrochloric acid.

6. The composition according to claim 5, wherein said acidifying agent is acetic acid or lactic acid.

7. The composition according to any one of claims 1 to 6, wherein said CsNWs are present in an amount of between 1 and 100 mg per mL of the composition, preferably between 20 and 70 mg / mL, more preferably equal to 25, 35 or 50 mg per mL of the composition.

8. The composition according to any one of claims 1 to 7, wherein said plasticizer is present in an amount of between 2% and 50% by weight with respect to the total weight of said CsNWs in the composition, preferably equal to 30% by weight.

9. The composition according to any one of claims 1 to 8, wherein said at least one polyphenolic compound is selected from tannins, lignin, or derivatives thereof.

10. The composition according to any one of claims 1 to 9, wherein said at least one polyphenolic compound is selected from tannic acid (TAN), epigallocatechin gallate (EPCG), mimosa tannin (MIM), lignosulfonates such as hardwood lignosulfonates (HLS), in particular methanol insoluble hardwood lignosulfonate fraction (MIHLS), Kraft lignin (KL), in particular ethanol insoluble Kraft lignin fraction, lignin nanoparticles or combinations thereof.

11. The composition according to any one of claims 1 to 10, wherein said at least onepolyphenolic compound is present in an amount of between 1% and 40% by weight with respect to the total weight of said CsNWs in the composition.

12. The composition according to any one of claims 1 to 11 , wherein said aqueous solvent is water or a mixture of water and ethanol, preferably wherein the ratio of water to ethanol in said mixture ranges from 1 to 3 v / v.

13. The composition according to any one of claims 1 to 12, further comprising at least one conductive compound selected from biochar, reduced graphene oxide, graphene, and carbon dots.

14. The composition according to any one of claims 1 to 13, further comprising at least one pigment or chromophore such as indigo carmine or rosa bengala.

15. The composition according to any one of claims 1 to 14, further comprising a photodegradable compound, such as methylene blue.

16. The composition according to any one of claims 1 to 15 which is selected from:- an aqueous composition comprising CsNWs in an amount equal to 35 mg / mL, glycerol in an amount equal to 30% by weight with respect to the total weight of CsNWs, tannic acid in an amount ranging from 1% to 30% by weight with respect to the total weight of CsNWs, acetic acid and water;- an aqueous composition comprising CsNWs in an amount equal to 35 mg / mL, glycerol in an amount equal to 30% by weight with respect to the total weight of CsNWs, epigallocatechin gallate (EPCG) in an amount ranging from 1 % to 5% by weight with respect to the total weight of CsNWs, acetic acid, and water; and- an aqueous composition comprising CsNWs in an amount equal to 35 mg / mL, glycerol in an amount equal to 30% by weight with respect to the total weight of CsNWs, hardwood lignosulphonates in an amount ranging from 1% to 40% by weight with respect to the total weight of CsNWs, preferably between 1 and 20% by weight, acetic acid, and water.

17. The composition according to any one of claims 1 to 15, which composition comprises CsNWs in an amount equal to 25 mg / mL, glycerol in an amount equal to 30% by weight with respect to the total weight of CsNWs, lactic acid, and water.

18. The aqueous composition according to claim 17, wherein said composition has a pH of between 5.8 and 6.1.

19. The aqueous composition according to any one of claims 1 to 18, in the form of a spray.

20. The aqueous composition according to any one of claims 1 to 15, which composition comprises CsNWs in an amount equal to 50 mg / mL, glycerol in an amount equal to 30% by weight with respect to the total weight of CsNWs, a pigment or chromophore such as indigo carmine, rosa bengala, zinc oxide, acetic acid and an aqueous solvent selected from water or a mixture of water with ethanol, preferably in a ratio of 1 , 2 or 3 v / v.

21. The composition according to the preceding claim, in the form of a nail polish composition.

22. The composition according to any one of claims 1 to 21 , wherein said composition is a colloidal suspension.

23. The composition according to any one of claims 1 to 22, wherein said CsNWs are prepared by surface deacetylation of chitin nanowhiskers (CtNWs) with NaOH under reflux and mechanical stirring.

24. A process for preparing a composition according to any one of claims 1 to 23, comprising at least the following steps:(i) providing a suspension of deacetylated chitin nanowhiskers (CsNWs);(ii) dispersing said suspension into an aqueous solvent;(iii) adding an acidifying agent to the dispersed suspension, preferably to reach a pH value of between 4 and 6.5, and(iv) preferably adding, in any one of the above-mentioned steps, a plasticizer; wherein said process further comprises adding, in any of the steps (i) to (iii), at least one polyphenolic compound.

25. The process according to the preceding claim, wherein said step (i) comprises subjecting a suspension of chitin nanowhiskers (CtNWs) to surface deacetylation with NaOH under reflux and mechanical stirring.

26. The process according to claim 25, wherein mechanical stirring is performed for 12 hours.

27. The process according to any one of claims 25 or 26, wherein, following deacetylation, theresulting suspension is subjected to a number of washing and centrifugation cycles until neutral pH is reached.

28. The process according to any one of claims 24 to 27, wherein said aqueous solvent is selected from water and a mixture of water and ethanol, preferably wherein the ratio water: ethanol in said mixture ranges from 1 to 3 v / v.

29. The process according to any one of claims 24 to 28, wherein, in said step (ii), said suspension of CsNWs is dispersed into said aqueous solvent to reach a final amount of CsNWs comprised between 1 and 100 mg / mL, preferably between 20 and 50 mg / mL, more preferably equal to 25 or 35 mg / mL.

30. The process according to any one of claims 24 to 29, wherein said step (ii) is performed by mechanical or magnetic stirring followed by ultrasonication.

31. The process according to any one of claims 24 to 30, wherein, in said step (iii), a solution of acetic acid 1.6 M is added until a pH in the range of 5.4 and 5.6 is reached.

32. The process according to any one of claims 24 to 31, wherein said at least one polyphenolic compound is selected from tannins, lignin, or derivatives thereof.

33. The process according to any one of claims 24 to 32, further comprising adding, in anyone of steps (i) to (iii), at least one conductive compound selected from biochar, reduced graphene oxide, graphene and carbon dots, and / or methylene blue and / or at least one pigment or chromophore such as indigo carmine or rosa bengala.

34. A process for preparing a film or coating comprising deacetylated chitin nanowhiskers (CsNWs), said process comprising the following steps:(i) applying a composition according to any one of claims 1 to 23 onto a target surface or substrate; and(ii) allowing the aqueous solvent of said composition to evaporate from the composition so as to form said film or coating.

35. The process according to claim 34, wherein said step (i) is carried out by solvent-casting, brushing, spraying, painting and / or rolling.

36. The process according to claims 34 or 35, wherein said step (i) is carried out by depositingsaid composition into one or more molds.

37. The process according to claim 36, wherein said one or more molds are circular or rectangular molds.

38. The process according to any one of claims 34 to 37, wherein said step (i) is carried out by applying an amount of said composition so as to form a film or coating that, after evaporation of said aqueous solvent, has a thickness comprised between 5 and 120 pm, preferably a film having a thickness comprised between 30 and 80 pm, more preferably equal to about 50 pm, or a coating having a thickness comprised between 5 and 20 pm.

39. The process according to any one of claims 34 to 38, wherein said step (ii) is carried out at room temperature and atmospheric pressure.

40. The process according to any one of claims 34 to 39, wherein said target surface or substrate is selected from plastic, paper, glass, fabric, tissue, polymeric material, plant material.41 . The process according to any one of claims 34 to 40, further comprising a step (iii) following step (ii) of separating the formed film or coating from said target surface or substrate.

42. A film or coating obtainable by a process according to any one of claims 34 to 41.

43. A composition according to any one of claims 1 to 23, for use as a medicament.

44. The composition for the use according to claim 43, in the treatment and / or prevention of skin conditions, in particular skin wounds, lesions, burns and / or abrasions.

45. A biomedical, sanitary, or cosmetic article comprising or consisting in a composition according to any one of claims 1 to 23 or a film or coating according to claim 42, said film or coating being optionally deposited or adsorbed onto a support.

46. The article according to claim 45, wherein said support is a polymeric support, a fabric, and / or an absorbent material.

47. The article according to claims 45 or 46, in form of bandages, gauzes, bands, protection films, patches, face mask each one as mono- or multi-layer.

48. The article according to claim 45, wherein said article is in the form of a spray.

49. A biomedical, sanitary, or cosmetic article in the form of a nail polish comprising deacetylated chitin nanowhiskers (CsNWs), an acidifying agent, and an aqueous solvent.

50. The article according to claim 49, comprising CsNWs in an amount equal to 50 mg / mL, glycerol in an amount equal to 30% by weight with respect to the total weight of CsNWs, a pigment or chromophore such as indigo carmine, rosa bengala, zinc oxide, acetic acid and an aqueous solvent selected from water or a mixture of water with ethanol, preferably in a ratio of 1 , 2 or 3 v / v.

51. A use of a composition comprising deacetylated chitin nanowhiskers (CsNWs), an acidifying agent, and an aqueous solvent for preventing or minimizing degradation of paper, preferably of ancient manuscripts, documents, or photographs.

52. The use according to claim 51 , wherein said composition is as defined in any one of claims 1 to 23.

53. A process for the fabrication of a film or coating onto a substrate for packaging of drugs, food and / or beverages, wherein said fabrication is carried out by means of a process according to any one of claims 34 to 41 .

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