EP3946281A1

EP3946281A1 - Polymer films comprising material secreted by gastropods

Info

Publication number: EP3946281A1
Application number: EP20714636.6A
Authority: EP
Inventors: Beatrice ALBERTINI; Nadia PASSERINI; Silvia Panzavolta; Luisa Stella Dolci
Original assignee: Universita di Bologna
Current assignee: Universita di Bologna
Priority date: 2019-04-02
Filing date: 2020-04-02
Publication date: 2022-02-09
Also published as: WO2020201439A1

Abstract

The present invention refers to a film, comprising at least one polymer and a material secreted by a gastropod, in particular snail slime. The present invention also relates to a procedure for the preparation and to uses of such film, in particular medical and cosmetic uses, including veterinary uses. Further objects of the present invention include kits, films for the storage and/or packaging of food, patches, masks and similar cosmetic products comprising such film.

Description

POLYMER FILMS COMPRISING MATERIAL SECRETED BY GASTROPODS FIELD OF THE INVENTION

BACKGROUND ART

The material secreted by snails includes numerous substances such as mucopolysaccharides, collagen, elastin, allantoin, glycolic acid, lactic acid, proteins, vitamins (vitamin A, E, C, B1 and B2), free amino acids, peptides, enzymes and trace minerals. Thanks to its characteristic composition, snail slime is known for its numerous advantageous properties, such as: cicatrizing, antibacterial, hydro-restorative, moisturizing, soothing, nourishing, regenerating, anti-wrinkle, anti-stretch mark, anti-acne, anti-stain. It is also capable of improving the appearance of scars and blemishes of various kinds (e.g. cellulite and stretch marks). In addition, the adhesive (J. Newar and A. Ghatak, Langmuir , 2015, 31, 12155-12160 and J. Li et ah, Science , 2017, 357, 378-381) and antimicrobial (G. Cilia and F. Fratini, J. Complementary Integr. Med., 2018, 20170168) properties of materials secreted by gastropods are known. It has also been reported that in in vitro fibroblast assays, extracts of Helix aspersa Muller are not cytotoxic, protect cells from apoptosis and induce cell proliferation and migration (C. Trapella et ak, Scientific Reports , 2018, 8, 17665).

These properties have been extensively exploited in the cosmetic and pharmaceutical fields. As a matter of fact, several snail slime cosmetic products are commercially available, e.g. in the form of creams and masks. In particular, hydrogel -based masks (jelly sheet masks), which have a gelatinous appearance and are often soaked in active elements, are known. They are disadvantageous, as they can leak once applied to the skin. Additionally, the presence of liquid prevents night-time use. In fact, they are usually masks that should be left on the skin for no more than 20 minutes. These products still employ a nonwoven fabric as a support.

Specific examples of commercially available snail slime products include:“Maschera Viso BIO con Acido Ialuronico e Aloe Vera” by Helidermina (http://www.helidermina.com/) and“Snail Jelly Mask” by SKEDERM (http://skederm.com/product/skederm-snail-jelly- mask-10-sheets-hydrogel-coated- facial-mask-with-snail-secretion-filtrate-5000ppm/).

In the pharmaceutical field, a syrup suitable for all bronchial airway irritation processes is also known.

In addition, the properties of snail slime have been the subject of several patent and scientific publications. For example, US patent 5,538,740 discloses an active ingredient extracted from snail useful for the therapeutic and cosmetic treatment of skin and mucosa, as well as creams comprising the same. Patent applications US 2017/0281690 A1 and US 2017/0216368 A1 describe the use of snail slime in liquid formulation in the treatment of rosacea and psoriasis, while patent application US 2013/0309296 A1 describes a device (gauze type) based on snail slime, for the prevention and treatment of diabetic ulcers and burns.

Patent application US 2010/0233111 A1 relates to cosmetic and pharmaceutical formulations comprising a biological fluid collected from gastropods (including in particular Helix Aspersa Muller) and methods for their preparation/production. They are useful for skin care. Such document further describes a method of collecting and refining biological fluid from gastropods. However, such document does not relate to compositions in the form of a film.

Patent application WO 2016/069396 A2 discloses a polymer film suitable for use as a face mask or transdermal patch, containing at least 30 wt. % of a thermoplastic polyurethane polymer and up to 5 wt. % of water. Such a film optionally comprises an active agent, which may be, among others, an agent that stimulates or regulates keratinocyte differentiation. Among these agents that stimulate or regulate keratinocyte differentiation are the gly coconjugates of Helix Aspersa Muller.

J. Li et al. (J. Li et ah, Science , 2017, 357, 378-381) developed resistant adhesives (“Tough Adhesives”, TAs) composed of two layers. The first layer is an adhesive surface comprising a positively interpenetrated polymer, while the second layer is a hydrogel- based dissipative matrix. Although the design of such resistant adhesives is inspired by the structure of the mucus secreted by Avion subfuscus slug, they do not contain any material secreted by a gastropod.

D. E. Lopez Angulo et al. and P. J. Do Amaral Sobral (D. E. Lopez Angulo et al. and P. J. Do Amaral Sobral, International Journal of Biological Macromolecules , 2016, 92, 645- 653) describe biologically active scaffolds comprising a mixture of gelatine, chitosan, aloe vera and snail mucus.

A. S. Haiti et al. (A. S. Haiti et al., Int. ./. Pharma Med. Biol. Sci., 2016, 5, 1, 76-80) report that administration of snail slime and chitosan is effective in wound healing.

However, the products described and/or marketed so far have several drawbacks. For example, skin and mucosal adhesion is suboptimal. This implies that the residence time of the active substances is shorter than necessary and that the product must be administered several times. Furthermore, the use of semi-solid formulations for topical administration of drugs, although very common and widespread, is associated with some drawbacks, such as uncertainty of the applied dose, the treated area and the contact time.

Therefore, there is still the need to develop a product comprising a material secreted by a gastropod that is adhesive and exhibits mechanical properties (e.g. elasticity and plasticity) useful for applying the product on skin, mucous membranes and/or nails.

DESCRIPTION OF THE INVENTION

The present invention relates to the addition of a material secreted by a gastropod, particularly snail slime, in liquid or lyophilised form, to formulations in the form of a film. The presence of the material secreted by a gastropod, preferably snail slime, allows to obtain films with unexpected properties, such as high flexibility, elasticity and bioadhesiveness. Accordingly, the films of the invention possess considerable advantages for applications in the cosmetic, pharmaceutical and veterinary fields as detailed below. As a matter of fact, depending on the disease of interest, the films can be loaded with active molecules. For example, films loaded with an antifungal agent (fluconazole) were effective at decreasing the spread of several fungi as assessed by the standardized Kirby-Bauer (KB) diffusion test. Moreover, permeation studies carried out on such antifungal agent-loaded films have revealed that the antifungal agent penetrates a skin layer.

In fact, the authors have developed films based on polymers, in particular natural, semisynthetic and/or biodegradable polymers, comprising a material secreted by a gastropod, preferably snail slime, in various concentrations for cosmetic, pharmaceutical and veterinary applications. In particular, the authors focused on the development of the formulation and on the preparation of such films. They may be prepared by solvent casting and may comprise further agents, in particular excipients, cosmetic agents and/or therapeutic agents. The films according to the present invention are characterized by numerous advantageous properties. For example, tensile tests revealed that they are endowed with desirable mechanical properties, such as high extensibility, stretchability, flexibility, deformability and manageability and at the same time reduced/low stiffness, rigidity and fragility. In addition, such films exhibit both elastic and plastic properties. At the same time, the films exhibit a bioadhesive behaviour, without requiring the addition of an adhesive agent.

The obtained films are bioadhesive, extensible, plastic, flexible. In particular, due to the bioadhesiveness of the films conferred by the material secreted by a gastropod, the addition of adhesive polymers may be unnecessary depending on the type of formulation. Further, the films of the present invention are neither rigid nor fragile. Therefore, they can be easily elongated and/or subjected to multiple stresses without breaking and exerting a light force. This makes them extremely versatile and adaptable.

As to adhesiveness, the films of the invention have proved effective at adhering to the surface of application, e.g. skin, nails, mucosa (e.g. gingival) and lips for prolonged periods of time (e.g. multiple hours), even when applied to areas subjected to frequent movement (e.g. palm of the hand, groove of arm).

Tack tests revealed excellent adhesive capacities, evidenced in particular by film detachment (F) and work of adhesion (W) values equal to or greater than 1 N and equal to or greater than 3 mJ, respectively, even on different surface types, and by an area under the plunger-raising speed vs time curve of 9 mJ or higher. Advantageously, films of the invention may have a transparency value measured as described below lower than 5 and a T% measured as described below equal to zero at 200-280 nm, suggesting that they may be used as sunscreen, e.g. to store food.

The films of the invention are characterised by a low water vapour permeability. It has also been found that film solubility and swelling degree and water vapour permeability may be controlled by changing film composition, so that films may be tailored to the intended use and a broad field of use can be covered. For instance, the films of the invention may act as fast dissolving films. The solubility of the films in water varies from complete solubility in a few minutes to insolubility for several months.

Thickness of the films may also be tailored to the specific need. Preferably, films of the invention have a thickness from 30 pm to 350 pm or from 50 pm to 350 pm. Variations in composition can be utilized to modulate the properties of the films for possible applications including those for the biomedical field or as edible coating for food packaging.

In vitro assays in epithelial cells and in fibroblasts have shown that the films of the invention do not interfere with cell proliferation or cell viability. Moreover, they do not cause Lactate Dehydrogenase (LDH) release, which indicates that they are not cytotoxic. Overall, films of the invention possess a promising safety profile. Additionally, they stimulate Fibroblast Growth Factor (FGF) and Collagen type 1 (COL1) release/production, i.e. in treated fibroblasts, there is a higher level/amount of FGF and COL1 than in untreated fibroblasts.

The presence of a material secreted by a gastropod confers to the films the properties of such material, such as for example cicatrizing, antibacterial, moisturizing, soothing, nourishing, regenerating, anti-wrinkle, anti-cellulite, anti-stretch marks, anti-acne, anti stain, improving the appearance of scars and blemishes of various kinds. In particular, the material acts like a plasticizer enhancing films extensibility and strongly improving their water barrier and bioadhesion properties, with a trend depending on material’s content. Furthermore, the material provides the films with antibacterial properties and enhanced cytocompatibility, yielding materials with tailored properties for specific requirements. The films of the invention have also proved effective at reducing the spread of a variety of Gram-negative and Gram-positive bacteria.

Such properties make the films of the invention particularly suitable for application to the human or animal body, in particular to the skin, mucous membranes, gums and nails. Films of the invention can advantageously be applied to areas of the body most susceptible to movement/bending, such as for example fingers, hands, wrists, ulnar cable, popliteal cable, ankle, armpit, feet.

In particular, the presence of snail slime in the films, alongside its aforementioned functional properties, provides the polymeric materials that are commonly used in the preparation of films high elastic and plastic properties (plasticizing effect), as well as a high adhesiveness (bioadhesive). The films object of the invention can remain adhered to the skin for more than 8 hours without the need to add adhesive polymers and can be easily removed by washing with water. In particular, the films produced can be applied by placing them in contact with a minimum amount of water to promote adhesiveness (non patch patch concept). Alternatively, appropriately formulated films could be used simply by wetting the skin with water and dissolving the film directly onto the skin. In this way a gelatinous film is formed as if it were a semi-solid formulation for skin application with moisturising (cosmetic) action.

The films can be prepared as a monolayer or multilayer (one-layer or multiple layers) with varying thickness depending on the application and can be loaded with a single drug or a combination of drugs. The various layers could comprise different polymers depending on the application and type of active molecules inserted. In particular, the films of the invention may be formulated as a composition in which two or more layers of different composition are assembled, thus obtained from films consisting of different polymers so as to modulate the release properties of the inserted active substances.

Therefore, if applied to skin or nails, they may be slightly wetted, while if applied topically they adhere perfectly to wet mucosal membranes (buccal, vaginal...). When loaded with active ingredients, the purpose is for instance to release the active ingredient locally (skin, oral/vaginal cavity, nails).

In addition, the films are perfectly transparent and adaptable to any shape of the body without the need to add plasticizers (additives commonly used in the formulation of films) and preservatives (bactericides and/or bacteriostats).

In the present invention, it is possible to use in the film a material secreted by a gastropod (in particular snail slime) as such (fresh or pure) or in dry (lyophilised or freeze-dried) form, thus preserving bioactivity, increasing preservability over time and facilitating the storage of said material. Lyophilization of said material can be accomplished by a standard procedure, for example using cryoprotectants such as dextran. Advantageously, the material’s bioactivity can be preserved over time by freeze-drying it.

The polymers may be used at different % w/V according to their molecular weight.

Therefore, the films object of the present invention represent a very versatile innovative system thanks to the mechanical, chemical -physical and functional properties linked to the presence of a material secreted by a gastropod, in particular snail slime, thus useful in the pharmaceutical, veterinary and cosmetic fields.

In particular, in the cosmetic field, the following applications can be identified: moisturizing, soothing, cellulite treatment (for example with possible addition of caffeine), such as anti-wrinkle or stain remover, thanks to the exfoliating properties of secretion and for the treatment of onychophagia following the addition of substances with an unpleasant taste (currently there are enamels/solutions containing molecules such as denatonium benzoate, or plant extracts with a strong bitter and spicy taste such as gentian or rhubarb). With regard to the pharmaceutical field, the films can be drug delivery systems for both skin and mucosal application. In the first case they can be applied for the treatment of dermatological disorders, such as skin infections (viral such as herpes, bacterial or fungal), atopic dermatitis, acne vulgaris and psoriasis and for the treatment of nail infections, such as onychomycosis. A further area of application also concerns the treatment of wounds (wound healing), for which films containing slime could be very beneficial.

For mucosal application, the produced films represent suitable systems for the treatment of aphthas (for which aloe-based gels, mouthwashes, sprays and patches are now used), vaginal and buccal infections for the local release of antibacterials, antifungals, antiprotozoal s and antivirals.

As for the human use, also in the veterinary field, films loaded with appropriate medication (anaesthetic, antiseptic, antibacterial, etc.), can be used for the treatment of skin wounds (wound healing) and skin infections. By acting as a second skin, they eliminate or otherwise reduce the problem of bandages not well tolerated by animals, keeping the wound clean and limiting contact with parasites that prevent wound healing.

The films of the invention, in particular when suitably formulated with snail slime and biopolymers, can be used as food wrapper/packaging or to separate food components and also to improve the quality of food products thanks to the antibacterial activity of the films themselves. To support this application, see for example the following references: Hongxia Wang, Jun Qian, and Fuyuan Ding, Emerging Chitosan-Based Films for Food Packaging Applications, J. Agric. Food Chem. 2018, 66, 395-413; M. N. Antoniewski & S. A. Barringer Meat Shelf-life and Extension using Collagen/Gelatin Coatings: A Review, Critical Reviews in Food Science and Nutrition, 2010, 50:7, 644-653, DOI: 10.1080/10408390802606691; Han and Wang, Sodium alginate/carboxymethyl cellulose films containing pyrogallic acid: physical and antibacterial properties J Sci Food Agric, 2017, 97, 1295-1301. Such references do not describe the use of a material secreted by a gastropod but include examples of polymers that can be employed in the films of the invention.

Therefore, it is an object of the present invention a film comprising at least one polymer and a material secreted by a gastropod, wherein said material is slime, mucus, or gastropod extract, and wherein said material is fresh or lyophilised.

Preferably, the film is solid and/or lacks a backing sheet and/or exhibits both elastic and plastic properties. Elastic and plastic properties may be measured by any method known in the art to assess them. In particular, elastic and plastic properties may be evaluated by stress-strain curves, in turn obtainable for example from tensile tests (e.g. constant speed tensile test) which may be performed as described in Example 14 below. Preferably, the film exhibits elastic properties when, in the elastic part of the respective stress-strain curve, stress and strain are proportional and once the load is removed, the film recovers its original shape. Preferably, the film exhibits plastic properties when it does not recover its original shape when the load is removed. Preferably, the films improve their elastic properties when the amount of snail slime is high: see for example Figure 4 A II, stress strain curve referred to CS 3070 L where the material maintains its elastic properties until it breaks (over 50% of deformation). Films containing low snail slime content (see for example Figure 4 A II, stress strain curve referred to CS 7030), show both elastic and plastic properties: the elastic region is limited to strain values less than 5%, after which the deformation mode is plastic (films do not recover their original length even if the load is removed).

Depending on the formulation design, the applied solid film can act as a fast dissolving film, a mucoadhesive film and as an adhesive soluble or insoluble film.

Preferably, said polymer is natural, semi synthetic, synthetic and/or biodegradable. Preferably, said polymer is selected from the group consisting of: gelatine, chitosan, cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, an alginate salt, carboxyvinyl polymer, rubber, carrageenan, a hyaluronate salt, starch, keratin, acrylic polymer and a combination thereof.

Preferably, said gelatine is porcine gelatine, bovine gelatine or fish gelatine.

Preferably said cellulose is selected from the group consisting of: hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose.

Preferably, said alginate salt is sodium alginate.

Preferably, said rubber is xanthan, guar or arabic rubber.

Preferably, said acrylic polymer is a polyacrylic acid or a methylmethacrylate.

Preferably, said combination is a combination of a cellulose and a hyaluronate salt.

In a preferred form, said polymer is cross-linked. Preferably, said polymer is cross-linked with an agent that is of natural origin and non-toxic. Preferably, said polymer is cross- linked with any one of: the material secreted by a gastropod, a hyaluronate salt, a further polymer with opposite charge to said polymer, citric acid, gallic acid, tannic acid or ferulic acid. It is to be understood that cross-linking of the polymer directly with the material secreted by a gastropod takes place depending on the polymer type and/or the ratio between the polymer and the material secreted by a gastropod. In a particularly preferred form, the invention provides a film as defined above wherein said polymer is gelatine, chitosan, cellulose, an alginate salt or a hyaluronate salt and is cross-linked with an agent that is of natural origin and non-toxic, preferably with a hyaluronate salt, citric acid, gallic acid or ferulic acid. Preferably, said polymer is cross-linked without using a cross-linking agent. For example, using 2% w/V chitosan, solubilized in 2% v/v acetic acid solution, in a volume ratio (chitosan : snail slime) 70 : 30, the material is insoluble in water for several weeks. A cross-linked film is thus obtained without using other chemical components. The use of the material secreted by a gastropod, in particular snail slime, can lead to cross- linked films. The presence of cross-linking may be evaluated by any method known in the art. For example, it may be evaluated by measuring the solubility of the film, wherein a film that is insoluble in water for several weeks comprises a cross-linked polymer.

Preferably, said hyaluronate salt is sodium hyaluronate.

Preferably, said gastropod is a slug or a snail. Still preferably, said gastropod is selected from the group consisting of: Helix aspersa, HelixComplex , Helix Pomatia, Helix Vermiculata, Helix aperta, Helix albescens, Helix ceratina, Helix engaddenis, Helix godetiana, Helix lucorum Linnaeus, Helix lutescens, Helix melanostomata, Helix obruta, Helix pomatia, Helix texta, Cornu Aspersum, Theba pisana, Otala Lactea, Cernuella virgata, Capaea, Euglandina rosea , Achatina fulica, Helix lucorum, Rapana venosa, Macrochlamys indica and Arion subfuscus.

In a particularly preferred form, said material secreted by a gastropod is snail slime.

In a further preferred form, the film of the invention further comprises:

- one or more excipients, and/or

- a cosmetic and/or therapeutic agent, and/or

- a micro/nanoparticulate system.

Preferably, the film as defined above comprises nanoparticles for modulating the permeability and/or other micro/nanoparticle systems to modulate the release of the active substance and increase skin permeability. Also, preferably, said micro/nanoparticulate system comprises or consists of mica, clay, or montmorillonite particles.

Preferably, said cosmetic and/or therapeutic agent is selected from the group consisting of: antifungal agent, antibacterial agent, antiprotozoal agent, antiviral agent, keratolytic agent, exfoliating agent, anti-inflammatory agent, analgesic agent, anaesthetic agent, antiseptic agent, antihistamine agent, anti-scabies agent, antioxidant agent, a proteolytic enzyme, natural alkaloid, agent for the treatment of onychophagy and agent that stimulates the proliferation of fibroblasts. In the present invention, “agent for the treatment of onychophagy” refers to a substance with an unpleasant taste, for example bitter and/or spicy. For example, an agent for the treatment of onychophagy is an enamel and/or a solution comprising denatonium benzoate. Another example of an agent for the treatment onychophagy according to the present invention is a plant extract with a strong bitter and/or spicy taste, such as for example a gentian or rhubarb extract.

Still preferably, said antifungal agent is an antimycotic of the azole and imidazole drug family.

Even more preferably, said cosmetic and/or therapeutic agent is selected from the group consisting of: fluconazole, econazole, 1,3,7-trimethylxanthine, sodium hyaluronate, citric acid, glycerol, miconazole, ketoconazole, clotrimazole, itraconazole, terbinafme, lidocaine, procaine, xylocaine, lidocaine hydrochloride, cortisone, cortisone derivative, promethazine, diphenhydramine hydrochloride, desclofeniramine maleate, catalase, erythromycin, tetracycline, gentamicin, neomycin, bacitracin, silver sulfadiazine, silver salt, chlorhexidine, chloramphenicol, thymol, acyclovir, permethrin, salicylic acid, diclofenac, ibuprofen, tea extract, aloe extract, tea tree oil, Opuntia Ficus Indica extract, vitamin, amino acid, cysteine, glycine, teronine, hyaluronate, growth factors, an alkyl- gallate and a tocopherol.

Preferably, said cortisone derivative is hydrocortisone acetate or triamcinolone acetonide. Preferably, said vitamin is selected from the group consisting of vitamin A, vitamin D, vitamin E, vitamin K and a mixture thereof.

Preferably, the film of the invention comprises an antifungal agent such as fluconazole.

In a further preferred form, the film of the invention is characterized by any one or more of the following parameters:

- stress at break (o_b) from 0.1 to 100 MPa (preferably from 0.5 to 95 MPa or from 0.2 to 60 MPa),

- elongation at break (e_b) from 1 to 2000% (preferably from 1 to 150% or from 8 to 200%),

- Young modulus (E) from 0.1 to 6000 MPa (preferably from 0.1 to 4500 MPa or from 0.2 to 2500 MPa), - maximum stress (o_m) from 0.1 to 100 MPa (preferably from 0.5 to 95 MPa or from 0.1 to 60 MPa),

- detachment force (F) equal to or higher than 1 N (preferably equal to or higher than 2 N or than 3 N or than 4 N, preferably from 4 to 20 N or from 4 to 18 N),

- transparency equal to or lower than 5, wherein transparency is measured as:

wherein Tί,oo is the transmittance at 600 nm and X is the thickness of the film,

- a value of T% measured between 200 and 280 nm equal to zero, wherein T% = f/Io x 100, where h = light transmitted from the sample and Io = light transmitted from the source or incident light,

- an X-ray diffraction pattern comprising a broad halo at 20 from 15° to 25° measured using CuKa radiation (optionally 40 mA, 40 kV, and 1.5 A), and/or

a water vapour permeability (WVP) measured as described in Jian-Hua Li et ah, Food Hydrocolloids 37 (2014) 166-173 or measured with the formulas defined in Example 23 below comprised between 10.00 x 10 ¹² and 1.50 x 10 ¹⁰ g m/s m² Pa.

Preferably, said detachment force is defined as the force required to detach a film that has adhered to a surface, such as skin, glass or aluminum. Said detachment force can be measured with any method known in the art, for instance using a rheometer or a dynamometer, e.g. by applying the film to the surface with a force of 5 N for 30 s, then raising the plunger at 1 mm/s.

It is to be understood that these parameters depend on several factors, such as polymer used and its amount, degree of drying of the film, storage time, etc. Further, it is to be understood that the polymer of the invention may be characterized by any combination of such parameters. Additionally, it is to be understood that any method known in the art may be used to measure such parameters, the methods described in the Examples being preferably used.

Preferably, the film does not alter cell viability or cell proliferation compared to a proper control, such as untreated cells. Cell viability and proliferation may be measured according to any known method, in particular those described in the Examples below.

Preferably, the film is not cytotoxic. Cytotoxicity may be measured according to any known method, for example by measuring the production/release of lactate dehydrogenase (LDH) in vitro. Then, the film of the invention preferably does not induce/stimulate/cause LDH production compared to a proper control, such as untreated cells or cells treated with a known cytotoxic agent such as phenol.

Preferably, the film improves wound healing. Improvement of wound healing may be measured by any known method, for instance in vitro by the production/release of fibroblast growth factor (FGF) and/or collagen type 1 (COL1) by cells. The film of the invention preferably increases FGF and/or COL1 production compared to a proper control, such as untreated cells or cells treated with a known cytotoxic agent such as phenol.

Preferably, the film is antibacterial, particularly effective against Gram-positive and/or Gram-negative bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Antibacterial effect may be measured by any known method, including the Kirby-Bauer (KB) diffusion test, e.g. as described in EUCAST: The European Committee on Antimicrobial Susceptibility Testing, Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 6.0, 2016. http://www.eucast.org. In particular, the antibacterial effect may be measured as described below. Preferably, the film is said to be antibacterial when, after an appropriate incubation time of for example about 24 h at 25 to 40 °C in a Mueller-Hinton agar plate pre-loaded with a bacterium, a halo appears around the film. Preferably, any one or more of the agents in the film penetrates the surface on which the film is adhered, such as skin, mucosa, nail, lip, etc. Surface penetration may be measured by any known method, e.g. using a Franz Cell system, such as by a permeation study performed as described in the Examples below.

It is a further object of the present invention a procedure to prepare a film as defined above, for instance by solvent casting.

An object of the present invention is a procedure for the preparation of a film comprising the steps of:

al) mixing at least one polymer dissolved in a solvent and a material secreted by a gastropod to obtain a solution, or

a2) mixing at least one polymer directly in a material secreted by a gastropod, and optionally successively adding a solvent to obtain a solution, and

b) evaporating said solution to obtain a film,

wherein said material is slime, mucus, or gastropod extract and wherein said material is fresh or lyophilised. In particular, an object of the present invention is a procedure for the preparation of a film comprising the steps of:

al) mixing at least one polymer dissolved in a solvent and a material secreted by a gastropod to obtain a solution, wherein said material is fresh, or

a2) mixing at least one polymer directly in a material secreted by a gastropod, and optionally successively adding a solvent to obtain a solution, wherein said material is fresh, or

a3) mixing at least one polymer dissolved in a solvent and a material secreted by a gastropod dissolved in water to obtain a solution, wherein said material is lyophilised, and b) evaporating said solution to obtain a film,

wherein said material is slime, mucus or gastropod extract.

Preferably, said step b) is performed by leaving said solution to evaporate or by heating it. Preferably, the total volume of said solution is comprised between 10 ml and 30 ml or between 15 ml and 25 ml. Preferably, the volume of said solution is of approximately 20 ml.

Preferably, any one of said procedures comprises a further step c) after step al) or a2) or a3) and before step b), said further step c) being: pouring said solution in a Petri dish. Preferably, the diameter of the Petri dish is comprised between 2 cm and 10 cm, between 5 cm and 9 cm or between 5.5 cm and 8.5 cm. Preferably, the volume of solution that is poured in the Petri dish is comprised between 5 and 10 mL, preferably it is of approximately 7.4 mL. Preferably, the weight of solution that is poured in the Petri dish is comprised between 5 and 15 mL, preferably it is of approximately 10.2 g.

Preferably, any one of said procedures comprises a further step d) after said step b), said further step d) being: depositing a cross-linking agent on the film. Preferably, said cross- linking agent is a 0.1% w/V aqueous solution of sodium hyaluronate.

Preferably, said solvent is water. Preferably, said water is acidified. Also, preferably, said solvent is acid. Still preferably, when said polymer is soluble in acidic environment, for example when said polymer is chitosan, said water is acidified. Also, preferably, when said polymer is gelatine, said water is not acidified. Preferably, said acidified water is acidified with hydrochloric acid, acetic acid, lactic acid or citric acid. For certain cosmetic applications, such as the exfoliating effect, an acidic pH of the solution is preferred, while for other skin applications and wound healing a pH > 6 is preferred. In particular, the pH of the material secreted by a gastropod and polymer solution can be determined and varied by adding an acid or a base before evaporation. Alternatively, it is possible to mix the polymer solution and the material secreted by a gastropod by controlling the pH of both in order to modulate the final pH.

In a preferred form of any one of said procedures, said polymer is in a concentration equal to or greater than 0.1% w/V with respect to the volume of said solution. In a preferred form of any one of said procedures, said polymer is in a concentration equal to or lower than 45% w/V or than 10% w/V or than 5% w/V with respect to the volume of said solution. In a further preferred form of any one of said procedures, said solution comprises from 5% to 100% in terms of volume of said material secreted by a gastropod. Preferably, in any one of said procedures, said solution comprises from 5% to 99.9%, from 20% to 80%, from 30% to 70%, from 40% to 60% or about 50% in terms of volume of said material secreted by a gastropod. Preferably, in any one of said procedures, said solution comprises approximately 100%, 70%, 60%, 50%, 40%, 30% or 15% in terms of volume of said material secreted by a gastropod. It is to be understood that the remaining volume of said solution comprises or consists of the polymer dissolved in the solvent. Likewise, it is to be understood that additional agents (e.g. an acid, a base, an excipient such as glycerol, etc.) can be added to the solution at any phase of the procedure prior to evaporation (step b), for instance to the polymer dissolved in the solvent, to the material secreted by a gastropod or to the obtained solution.

It is a further object of the invention the film obtainable by the procedure as described above. In particular, it is an object of the invention the film as defined above obtainable by the procedure as defined above.

In a preferred aspect, the film of the invention is obtainable from any one of the procedures defined above employing the following parameters:

It is to be understood that: the % w/V of polymer is reported as weight of polymer / total volume of solution; the % V/V of solvent is reported as V of solvent / total volume of solution; the % V/V of material secreted by a gastropod is reported as V of material secreted by a gastropod (when step al) or a2) was employed) or V of water (when step a3) was employed) / total volume of solution.

Preferably, the invention also provides the film obtainable by subjecting CS 7030 A, CS_3070_A or CS_3070_SOL to step d) as defined above using a 0.1% w/V aqueous solution of hyaluronate.

It is also an object of the invention the film as defined above for use as a medicament. Preferably, the film as defined above is for use in a method of preventing and/or treating: a dermatological disorder, a cutaneous wound, an aphtha, an infection, dermatitis, atopic dermatitis, radiotherapy dermatitis, eczema, rash, acne vulgaris, psoriasis, rosacea, a burn, a sunburn, an ulcer, a diabetic ulcer, a scald, onychomycosis, onychophagia and/or a periodontal disease. Preferably, preferably said infection is fungal, bacterial or viral. Preferably, said infection is a vaginal infection, a buccal infection, a skin infection, a nail infection, a mucosal infection, a lip infection, a wound infection. Preferably, said infection is selected from the group consisting of: a vaginal infection, a buccal infection, a skin infection, a nail infection, a viral infection of the skin, a viral infection of the mucosa, a viral infection of the lips, a bacterial infection of the skin, a bacterial infection of the mucosa, a fungal infection of the skin, an infection of a wound, a vaginal herpes, a lip herpes or cold sores.

Then, it is also an object of the present invention the film as defined above for use as an antibacterial, antifungal and/or antiviral agent. Preferably, said antibacterial agent is effective against Gram positive and/or a Gram negative bacteria, preferably, said bacteria are selected from the group consisting of: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and combinations thereof. Preferably, said antifungal agent is effective against a fungus of the Candida genus, including but not limited to: Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei.

The use of the film as defined above as a cosmetic product is also an object of the invention. The use of the film as defined above for packaging food is also an object of the invention. The use of the film as defined above for food storage is also an object of the invention.

A further object of the invention is a non-therapeutic cosmetic method for preventing and/or decreasing a skin imperfection (or skin blemish), hydrating/moisturizing the skin and/or soothing the skin or mucosa, said method comprising the administration and/or application of the film as defined above. Preferably said skin imperfection (or skin blemish) is cellulite, a stretch mark, a wrinkle, a scar, a stain and/or redness of the skin.

In a further aspect the present invention provides a kit comprising the film as defined above. In a further aspect, the present invention provides a wrap or foil comprising the film as defined above. In a preferred aspect said wrap or foil is a wrap or foil for the storage and/or packaging of food. Accordingly, the present invention provides the use of said wrap or foil for storing and/or packaging food. In a further aspect, the present invention provides a patch comprising the film as defined above. In a further aspect, the present invention provides a mask comprising the film as defined above. In a further aspect, the present invention provides a gauze comprising the film as defined above. In a further aspect, the present invention provides a drug delivery system comprising the film as defined above.

It is also an object of the present invention a system comprising at least two films, said films being as defined above. In particular, in one aspect, the present invention provides a system comprising at least a first film and a second film, said first film and said second film being as defined above. Preferably, the polymer in the first film is different from the polymer in the second film. Preferably, said first and said second films further comprise a cosmetic and/or therapeutic agent. Preferably, the cosmetic and/or therapeutic agent in the first film is different from the cosmetic and/or therapeutic agent in the second film.

It is a further object of the present invention a solution comprising a polymer and a material secreted by a gastropod, wherein said polymer is in a concentration equal to or greater than 0.1% w/V compared to the volume of said solution, wherein said material is slime, mucus, or gastropod extract, and wherein said material is fresh or lyophilised. It is a further object of the present invention a solution comprising a polymer and a material secreted by a gastropod, wherein said solution comprises from 5% to 100% in terms of volume of said material secreted by a gastropod, wherein said material is slime, mucus, or gastropod extract, and wherein said material is fresh or lyophilised. It is a further object of the present invention a solution comprising a polymer and a material secreted by a gastropod, wherein said polymer is in a concentration equal to or greater than 0.5% w/V compared to the volume of said solution, wherein said solution comprises from 5% to 100% in terms of volume of said material secreted by a gastropod, wherein said material is slime, mucus, or gastropod extract and wherein said material is fresh or lyophilised. Preferably, said solution comprises from 5% to 100% in terms of volume of said material secreted by a gastropod. Even more preferably, said solution comprises from 5% to 99.9%, from 20% to 80%, from 30% to 70%, from 40% to 60% or about 50% in terms of volume of said material secreted by a gastropod. Preferably, said solution comprises approximately 100%, 70%, 60%, 50%, 40%, 30% or 15% in terms of volume of said material secreted by a gastropod. It is to be understood that the remaining volume of said solution comprises or consists of the polymer dissolved in the solvent. Likewise, it is to be understood that additional agents (e.g. an acid, a base, an excipient such as glycerol, etc.) can be present in the solution.

The material secreted by the gastropod in pure (liquid) form can be used 100% (v/v): in this case the polymer (in powder form) and any other excipients are inserted directly into the material secreted by the gastropod, which can optionally be considered as the only solvent. Otherwise, the volume of the material secreted by a gastropod can be decreased and then mixed with a polymer solution at different v/v ratios (5:95 to 95:5, respectively). When the slime is used as a lyophilised powder it can be inserted into the preparation solvent from 5% to 100% w/V, in particular from 5% to 99.9%, preferably from 5% to 95% w/V.

It is a further object of the present invention the solution obtainable from step al), a2) or a3) of any one of the procedures as defined above. Preferably, said solution is obtainable employing the parameters reported in the table above (i.e. table describing the film of the invention obtainable from any one of the procedures defined).

In a further aspect the present invention provides a kit comprising the solution as defined above. In a further aspect, the present invention provides a wrap or foil comprising the solution as defined above. In a preferred aspect said wrap or foil is a wrap or foil for the storage and/or packaging of food. Accordingly, the present invention provides the use of said wrap or foil for storing and/or packaging food. In a further aspect the present invention provides a patch comprising the solution as defined above. In a further aspect, the present invention provides a mask comprising the solution as defined above. In a further aspect, the present invention provides a gauze comprising the solution as defined above. In a further aspect, the present invention provides a drug delivery system comprising the solution as defined above.

In a preferred embodiment, the film is administered to the skin, oral mucosa (particularly gums), vaginal mucosa, anal mucosa.

In a further preferred embodiment of the invention, the composition of the invention is administered to a human subject or an animal subject.

It is to be understood that the film of the invention is a composition comprising a polymer and a material secreted by a gastropod as defined above, said composition being in the form of a film.

Dosage forms for topical administration are generally classified as liquid, semisolid, and solid, as outlined in Figure 1 (taken from: Brown MB., Turner R., Lim ST. Topical product development, In: Transdermal and topical drug delivery. Principles and Practice. Edited by Benson HAE., Watkinson AC., Wiley 2012, pp. 255-286, herein incorporated by reference).

The evaporation of volatile components, the so-called vehicle metamorphosis (C. Surber, E.W. Smith, Dermatology , 210 (2) (2005), pp.157-168), may modify the characteristics of a semi-solid formulation and even precipitate the drug onto the surface of the skin. To overcome these drawbacks, a dermal patch or film may be an alternative (C. Padula et al. Eur. J Dermatol., 17(4) (2007), pp. 309-312).

Films can be classified as patches, but unlike medicated patches (plasters or cataplasms), they do not require, but may include, external support. These are therefore polymeric materials that can be applied directly to the skin (or mucosa or nail or elsewhere) with a light pressure. By definition, films per se do not have a support (backing sheet, which can be made of non-woven fabric or other non-occlusive or occlusive material). For example, transdermal patches normally consist of an outer covering which supports a preparation which contains the active substance(s). The outer covering is a backing sheet impermeable to the active substance(s) and normally impermeable to water, designed to support and protect the preparation. (European Pharmacopoeia 10 Ed.). However, the film components (meaning the solution/dispersion comprising polymer and material secreted by a gastropod before drying) can be coated on a support to obtain, for example, a medicated patch or a skin patch.

Films for topical use are generally thin, highly flexible dosage forms of a squared/rectangular shape and easily applicable even without the use of applicators. They are usually formulated with the active ingredient of interest together with water-soluble polymers, plasticisers (polyethylene glycol, glycerol, etc.), humectants (glycerol or other polyols), diluents, and/or additional substances that prevent microbial proliferation and/or increase the permeability of the drug into the skin and/or control its release over time. In the invention it is therefore possible to formulate films that solubilize and release the active ingredient and all functional components (bioactive polymers and material secreted by a gastropod) very quickly. For example, the films of the invention can be broken up thus releasing snail slime. In particular, once the film is adhered to the skin, it can remain as such for the required period of time releasing the active components contained therein, and eventually be washed. For example, when applied to mucous membranes the film solubilizes more or less quickly depending on the formulation. A further object of the invention are cross-linked polymers-based films that do not solubilize, which can be combined with rapidly solubilizing films in order to create a controlled release system. Films are solid but flexible dosage forms that adhere to the skin due to the presence of an adhesive substance and that, depending on the composition, can be removed at the end of application or dissolved more or less quickly in situ. They therefore have the advantage of greater patient compliance, increase of the residence time of the active substances reducing the frequency of application and, unlike gels and creams that must be administered several times a day, the films remain adhered for several hours, do not“get dirty” and do not “stick” to the surrounding environment.

A further feature of films is that, unlike medicated patches, they can be formulated as easily removable by washing the skin and this is an advantage when the film is applied to damaged skin. In a further aspect of the invention, the film may be applied under occlusive conditions, exploiting the occlusion enhancing effect on drug administration.

The films may also be applied to mucous membranes (e.g., oral, vaginal) by appropriately modifying the formulation to control the disintegration of the film.

The main advantages of films over other pharmaceutical forms for skin and mucosal application are: flexibility and adaptability to all types of surfaces,

instantaneous adhesion to the surface of the mucosa after application, both to prolong the contact time between the formulation containing one or more active substances and the surface to be treated, and to avoid the use of adhesives that are often painful and uncomfortable at the time of removal (as is the case of patches),

mechanical strength in order to maintain its integrity under stress, and

easy removal after washing or natural dissolution of the device in the application area.

Preferably, the at least one polymer as defined above is not toxic or irritating, does not release impurities, is wettable and tensile resistant. In fact, the type of polymer employed, and its molecular weight can greatly affect the properties of the films and their disintegration/solubilization time.

Chitosan is a linear polysaccharide composed of D-glucosamine and N-acetyl-D- glucosamine bound through b bonds. Chitosan is derived from chitin, which is mainly derived from shells of crustaceans and molluscs, but also from fungi. In the present invention, chitosan may be low, medium or high molecular weight and exhibit a deacetylation degree for example from 5 to 98%, preferably equal to or greater than 93%. In particular, the molecular weight of chitosan may be, for example, from 40000 to 500000 MW or of approximately 100 KDa.

Gelatine is a protein obtained from the processing of collagen. In a preferred embodiment, the gelatine is cross-linked with naturally occurring and non-toxic agents, such as for example gallic acid, ferulic acid, and citric acid. Gelatine can be classified by, for example, the Bloom index, which is a measure of the stiffness of the gel it forms under certain conditions. In the present invention, gelatine may have a Bloom index e.g., from 20 to 300 Bloom.

Regarding gelatine, the cross-linking degree is measured spectrophotometrically, after derivatization of the e-amino groups of lysines, present in the macromolecular chain, with an appropriate reagent (trinitro-benzenesulfonic acid) Ofner CMIII, Bubnis WA, Chemical and swelling evaluation of amino group crosslinking in gelatine and modified gelatine matrices. Phar. Res. 1996; 13: 1821-7.

Cellulose is a polysaccharide in which glucose units are bound by a b 1 4 glycosidic bond. Cellulose polymers are divided into subclasses with very different characteristics. All polymers belonging to such subclasses may be used in the films of the invention. Alternatively or in addition to the aforementioned polymers, films may comprise cellulose derivatives: hypromellose with different molecular weight and/or of different percentage content of methoxyl and/or hydroxypropyl groups (such as that used in the Examples); as well as any cellulose derivative, such as hydroxyethylcellulose, hydroxypropylcellulose, methyl cellulose, carboxymethylcellulose and sodium carboxymethylcellulose. In the present invention, the terms“hydroxypropylmethylcellulose” and“hypromellose” (HPMC) are used as synonymous. HPMC, depending on the percentage content of methoxylic and hydroxypropyl groups, is divided into three families: Methocel E (HPMC 2910, USP), Methocel F (HPMC 2906, USP) and Methocel K (HPMC 2208, USP). Another HPMC classification parameter is the viscosity of 2% w/V Methocel solutions in water, measured at 20 °C: thus Methocel E is designated as E5, El 5 or E50 because the relative solutions have viscosity values of 5 mPa s, 12-15 mPa s and 40-56 mPa s, respectively.

Starch consists of two families of homopolysaccharides, amylose and amylopectin. The first is comprised of glucose chains with alpha 1-4 bonds while the second is comprised of alpha 1-4 glucose chains with branches in alpha 1-6 bonds at the branching point. The relationship between these two families indicates the molecular weight.

Keratin is a polymer that can be extracted from wool.

Gastropods are a class of molluscs comprising more than 65000 species and include both snails (Helix) and slugs (Limax). Among the most common snails are those belonging to the family Helicidae , such as: Helix Pomatia and Helix Aspersa (the most common) as well as Helix Complex, Helix Vermiculata, Helix aperta, Helix albescens, Helix ceratina, Helix engaddenis, Helix godetiana, Helix lucorum Linnaeus, Helix lutescens, Helix melanostomata, Helix obruta, Helix pomatia, Helix texta, Cornu Aspersum, Theba pisana, and Otala Lactea (synonym Helix ahmarina or Helix lactea). Examples of gastropods also include Cernuella virgata, Capaea , and Euglandina rosea. In the present invention, any one or more of such exemplary gastropods may be used to obtain the material secreted by a gastropod.

The following invertebrate zoology manuals are references to the description of the Gastropoda class: Evolutionary Developmental Biology of Invertebrates vol 2 ED. Springer; The Invertebrates: Volume VI Mollusca I, di Libbie H. Hyman 1967 and Invertebrate Zoology of EE. Ruppert and RD. Barnes ED Saunders College Pub, International ed 1994 VI edition and are herein incorporated by reference. The material secreted by gastropods commonly called“snail slime” is produced by salivary glands (pedal glands) and the main molecules present therein are: allantoin, glycolic acid, collagen, elastin, exfoliating lactic acid, mucopolysaccharides (GAGs and without sulfur incorporation), vitamins A, C, E, B1 and B6, free amino acids, peptides, proteins, enzymes, molecules with antiprotease activity (from the Italian heliciculture institute website www.istitutodielicoltura.it). The composition and function of materials secreted by a gastropod are described in the following publications, herein incorporated by reference: G. Cilia and F. Fratini, J. Complementary Integr. Med., 2018, 20170168 and C. Trapella et al. Scientific Reports, 2018, 8, 176665.

Slime allows snails to move, and thanks to its adhesive properties it allows a movement even on vertical or particularly complex surfaces. Slime is also produced to keep the body of the gastropod lubricated, hydrated and moistened but also as a defence from predators. An extract of a gastropod refers to the entire body of a gastropod that has been blended. By “pure” or“fresh” material secreted by a gastropod, it is meant a material secreted by the gastropod, not further treated/processed. By “dry” or“lyophylised” or“freeze-dried” material secreted by a gastropod, it is meant that the material as secreted by the gastropod has been treated/processed, in particular by lyophilization.

The material secreted by a gastropod can be obtained by known techniques, for example as described in US 5,538,740, WO201311371 and IT 10207000117547, herein incorporated by reference. Particularly, the material secreted by a gastropod may be obtained by the Miiller method. Alternatively, the material secreted by a gastropod can be extracted with manual stimulation as indicated in the Helidermine products (http://www.helidermina.com/).

In particular, in the Examples reported below, different snail slimes were employed for the film preparation. The detailed compositions are described in the next section (Detailed Description of the Invention). The mucus from Helix Aspersa, extracted by MullerOne method (as decribed at https://istitutodielicicoltura.it/it/en/mullerone and at http://www.mullerone.com/it/en/extraction-process_applied to Helix aspersa Muller snails), was used in the compositions reported in Tables 1, 2, 3, 4 and 5; while the HelixComplex® snail mucus, extracted by HelixPharma srl (patent applications publications N. IT 10207000117547 and N. WO 2013011371 Al), was employed for the film compositions reported in Tables 5 and 6. The material secreted by a gastropod using the Miiller method was kindly supplied by Azienda Agricola I PODERI - Azienda Agricola - Elicicoltura, Poderi di Montemerano 58014 Manciano (GR).

After extraction using the Miiller method, snail slime can be stored until further use as such (pure or fresh) or after freeze-drying in the optional presence of a lyoprotectant such as dextran. Storage of the fresh or dried snail slime can be carried out at -25°C to 5°C.

According to the present invention, the material secreted by a gastropod, in particular snail slime, can be preferably characterised by the features reported in the following table.

where M.I.M. stands for Multiple Ion Monitoring mode.

In particular, the snail slime kindly donated by Azienda Agricola I PODERI and employed in most of the experiments below is characterised by the features in the table above.

Cosmetic and/or therapeutic agents, i.e. drugs, can be dissolved or dispersed in the films, and can be inserted singularly or in combination with each other. In particular, the following agents may be inserted into the films: fluconazole/econazole (antifungals of the azole and imidazole drug family), 1,3,7-trimethylxanthine (caffeine, natural alkaloid), lidocaine hydrochloride (local anaesthetic). Further agents that may be inserted into the films include: cortisone derivatives (e.g. hydrocortisone acetate, triamcinolone acetonide, etc.); antihistamines (e.g. promethazine, diphenhydramine hydrochloride, desclopheniramine maleate, etc.); proteolytic enzymes (e.g. catalase, etc.); antibacterials (e.g. erythromycin, tetracyclines, gentamicin, neomycin, bacitracin, silver sulfadiazine or other silver salts, chlorhexidine, chloramphenicol, thymol, etc.); antivirals (e.g. acyclovir, etc.); anti-scabies (e.g. permethrin, etc.); antifungals (e.g. fluconazole, econazole, miconazole, ketoconazole, clotrimazole, itraconazole, terbinafms, etc.); local anaesthetics (e.g. lidocaine, procaine, xylocaine, etc.); keratolytics (e.g. salicylic acid, etc.), anti inflammatories and analgesics (e.g. diclofenac, ibuprofen, etc.).

Other active substances of natural origin that may be inserted into the films of the invention include: tea extracts, aloe extracts, tea tree oil, Opuntia Ficus Indica extract, vitamins (e.g., Vitamin A, D, E, K); amino acids and other substances capable of stimulating fibroblast proliferation (e.g. cysteine, glycine, teronine, hyaluronate, growth factors).

Preferably, said excipient is selected from the group consisting of: plasticizer, diluent, humectant, pH modifier (e.g. an acidifier or a basic substance), absorption promoter, fragrance, solubilizer, emulsifier, antioxidant, dye, preservative, nanoparticle, microparticle, a humectant, and a combination thereof.

Still preferably said plasticizer is polyethylene glycol or glycerol or polyethylene oxide. Also preferably said humectant is glycerol, sorbitol, propylene glycol, or polyethylene glycol. Said pH modifier may be for example a 1M NaOH solution. Absorption promoters include, for example, glycols, alcohols, surfactants. Said emulsifier is preferably a non ionic surfactant or a polysaccharide. Said antioxidant can be an alkyl-gallate or a tocopherol, for example. In a preferred embodiment, said excipient is a nanoparticle for modulating film permeability or a micro/nanoparticulate system for modulating the release of the active substance and increasing skin permeability. In particular, said nanoparticle may be mica, clay or montmorillonite. Preferably, said excipient is glycol.

The present invention will be described by means of non-limiting examples, referring to the following figures:

Figure 1. Dosage forms for topical administration, taken from: Brown MB., Turner R., Lim ST. Topical product development, In: Transdermal and topical drug delivery. Principles and Practice. Edited by Benson HAE., Watkinson AC., Wiley 2012, pp. 255- 286.

Figure 2. INSTRON 4465 dynamometer, detail, film fixed between the clamps.

Figure 3. (A) Appearance of the prepared chitosan-based films. On the right scanning electron micrographs of some selected films are reported. Bar = 100 pm. (B) Image of a gelatine-based film containing snail slime in the 30:70 ratio. (C) Image of a cellulose- based film (HPMC E5) containing snail slime in the 30:70 ratio.

Figure 4. (A) Exemplary stress-strain curves recorded on chitosan films prepared in (I) acetic acid (A); (II) lactic acid (L) and (III) snail slime (SOL). (B) Exemplary stress-strain curves obtained for GB and GBS 3070 bovine gelatine films. (C) Exemplary stress-strain curves obtained for E5, E5S 7030 and E5S 3070 cellulose films. (D) Exemplary stress- strain curves obtained for CMC -based films (CMC, CMCS_3070 and CMCS_100 films). Figure 5. Adhesion of CS 3070 A film. (A) Application to the inside of the palm of the hand, at the joining of thumb and forefinger. Photo taken at the time of application. (B) Application to the inside of the palm of the hand, at the joining of thumb and forefinger. Photo taken 8 hours after application. (C) Application in the groove of the arm. Photo taken 2 hours after application. (D) Application on nail. Photo taken at the time of application. (E) Application to the lips. Photo taken 1 hour after application. (F) application to the buccal mucosa. Photograph taken at the time of application.

Figure 6. (A) An INSTRON 4465 dynamometer detail, during the detachment measurement of the CS 3070 A film applied to porcine skin. (B) Curves recorded during the tack test measurements performed on CS 3070 L films by means of the Rheometer AntonPaar. Each curve corresponds to a distinct sample. Figure 7. Adhesive properties of chitosan-based films (***p < 0.001, **p < 0.01, *p < 0.05). Statistical analysis was performed with Graph Pad Prism 4. One-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test was employed to assess statistical significance of the experimental conditions; statistically significant differences were determined at p < 0.05. F = film detachment and W = work of adhesion. Figure 8. Freeze-dried snail slime in the absence (left containers) and presence (right containers) of lyoprotectant (2% w/w dextran) and other substances such as glycerol and dextran.

Figure 9. Proliferation measurements obtained using VERO cells for films produced with: chitosan only (C_A in grey), chitosan: slime 30:70 (CS_30:70_A in black), chitosan solubilised directly in snail slime (CS_3070_SOL in white). Cell viability is expressed as percent proliferation relative to control cells (grown in normal medium).

Figure 10. Vero cell viability after 48 h of incubation with the media containing film components following disks dissolution. Data (mean values ± SD) are relative to the untreated control grown in normal medium (set to 100%).

Figure 11. Cytotoxicity and bioactivity evaluation in human normal skin fibroblasts. CTR- = DMEM only; CTR+ = DMEM + 0.05% toxic phenol solution, lighter bars = measurements after 24 hours of culture; darker bars = measurements after 72 h of culture. Statistical analysis is reported in the figure (*p<0.05, **p<0.005, ***p<0.0005) (A) Human fibroblast viability; RFU = relative fluorescence units; at 24h: **CTR+ vs C2S_7030_A and CTR-; 72h: **CTR+, vs C2S_7030_A, CTR- (B) toxicity measured as Lactate Dehydrogenase (LDH) release in supernatant; LDH490/655nm/Alamar blue = LDH concentration was spectrophotometrically read at 490 / 655 nm; 24h: **CTR+ vs C2S_7030_A and CTR-; 72h: *CTR+ vs C2S_7030_A and CTR- (C) Fibroblast Growth Factor (FGF) *C2S_7030_A vs CTR+ and CTR- (D) Collagen type 1 (COL1) production *C2S_7030_A vs CTR-.

Figure 12. Water vapor permeability (WVP) (***p < 0.001, **p < 0.01, *p < 0.05). Statistical analysis was performed with Graph Pad Prism 4. One-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test was employed to assess statistical significance of the experimental conditions; statistically significant differences were determined at p < 0.05. Figure 13. UV visible spectra collected on cellulose (E5)-based films containing different polymenslime ratios. T%= Ii/Io* 100, where Ii = trasmitted light (transmitted from the sample) and Io = incident light (transmitted from the source)

Figure 14. Apple slices wrapped with CMCS_3070 film. Photo taken 2 days after application.

Figure 15. Permeation of Fluconazole (FL) across porcine skin.“FL film” is a 5% w/V porcine gelatin-based film containing snail slime, glycerol (30 % w/w on the dry polymer) and FL (5% w/w with respect to the total dry mass of gelatin, glycerol and snail slime, where gelatine : snail slime volume ratio 30 : 70).“FL solution” is a control solution of FL 0,45% w/V in phosphate buffer supplemented with 20% w/V ethanol.

Figure 16. X-rays diffraction patterns (left) and corresponding IR spectra (right) of chitosan films and lyophilized solution. (A) left: X-rays diffraction patterns of Chitosan films prepared by dissolving chitosan in acetic acid and containing different amounts of Snail slime; right: infrared spectra acquired on the same samples (B) left: X-rays diffraction patterns of Chitosan films prepared by dissolving chitosan in lactic acid and containing different amounts of Snail slime; right: infrared spectra acquired on the same samples (C) left: X-rays diffraction patterns of Chitosan films prepared by dissolving chitosan directly into snail slime and containing different amounts of high purified water (HPW); right: infrared spectra acquired on the same samples (D) left: X-rays diffraction patterns collected on lyophilized snail slime and (right) corresponding infrared spectrum. Figure 17. Derivative of TGA (DTG) plots recorded on: (A) Chitosan films prepared by dissolving chitosan in lactic acid and containing different amounts of Snail slime (B) Chitosan films prepared by dissolving chitosan in acetic acid and containing different amounts of Snail slime (C) Chitosan films prepared by dissolving chitosan directly into snail slime and containing different amounts of high purified water (HPW) (D) Lyophilized snail slime

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations

A = acetic acid

CA = citric acid

AL = alginate sodium salt

C = chitosan 1% w/V

C2 = chitosan 2% w/V CMC = sodium carboxymethyl cellulose

E = hydroxypropyl methylcellulose (HPMC) Methocel

E5 and E50 = hydroxypropyl methylcellulose (HPMC) (Methocel E5 and E50)

FDS = freeze-dried snail slime

GB = bovine gelatin

Gly = glycerol

GlylO = glycerol 10% (V/V)

GP = porcine or swine gelatin

H = Hyaluronate sodium salt

HO.3 = Hyaluronate 0.3% w/V

HI = Hyaluronate 1% w/V

H2 = Hyaluronate 2% w/V

HPW = high purified water

L = lactic acid

S = snail mucus or snail slime

S 100 or SI 00 = product wherein the polymer has been directly solubilized in the 100% volume of the snail slime

SOL = product wherein the polymer has been directly solubilized in the snail slime and water has been subsequently added

Exemplary films of the invention are labelled using the following nomenclature. The first abbreviation indicates the polymer used, namely: chitosan (C), porcine gelatin (GP), bovine gelatin (GB), hydroxypropyl methylcellulose E5/E50 (E), sodium carboxymethyl cellulose (CMC) and/or sodium hyaluronate (H). The letter "S" indicates the presence of snail slime. The abbreviation "Gly" indicates the addition of glycerol, while the abbreviation "SOL" indicates that the polymer (e.g. chitosan) was dissolved directly in the snail slime and the mixture thus obtained was subsequently added with water. For the chitosan-based films, "HCl", "A", and "L" indicate, respectively, that the polymer is solubilized in an aqueous solution acidified with hydrochloric acid, acetic acid and lactic acid. "H" as a final letter and "CA" indicate crosslinking of the polymer with hyaluronate and citric acid, respectively. When a film contains snail slime (S or FDS), the ratio in terms of volume between polymer solution and slime is indicated. For example, GBS 7030 indicates that the film was obtained from a solution containing 70 parts by volume of an aqueous bovine gelatine solution and 30 parts by volume of snail slime. When the polymer is directly solubilized in the snail slime without the addition of water, the film is indicated with S_100. For example, films E5S_100, E50S_100, CMCS _100, HS_100, HS_100 Gly contain the polymer directly solubilized in the snail slime, which corresponds to the final volume.

Glycerol (Gly) has been added as percent in weight (w/w, weight of glycerol with respect to the weight of dry polymer) or as a percent in volume (V/V, volume of glycerol with respect to the total volume of the solution).

Compositions of exemplary films

Tables 1-6 show the compositions of some films, as examples of the present invention. In particular, the films were prepared using the following polymers: chitosan (C), chitosan (C) cross-linked with sodium hyaluronate (H), porcine gelatine (GP), bovine gelatine (GB), porcine gelatine (GP) cross-linked with citric acid (CA), bovine gelatine (GB) cross-linked with citric acid (CA), hydroxypropylmethylcellulose (E), sodium carboxymethyl cellulose (CMC), sodim alginate (AL), sodium hyaluronate (H) and finally mixtures of CMC and H. Such polymers may be used at different concentrations (% w/V with respect to the total volume of the polymer-slime solution) relatively to the molecular weight of the polymer itself.

In all cases, the snail slime (S) was used at different volumes to obtain different ratios of polymer/slime solution (V/V).

Table 1 : Compositions of chitosan-based films (1 and 2% w/V)

Furthermore, using 1% w/V chitosan, solubilized in 2% v/v acetic acid solution in a chitosan : snail slime volume ratio of 85 : 15, this material (CS_8515_A) is insoluble in water for several weeks. Using 2% w/V chitosan, solubilized in 2% v/v acetic acid solution, in a chitosan : snail slime volume ratio of 70: 30, this material (C2S_7030_A) is insoluble in water for several months. A cross-linked film is thus obtained without using other chemical components.

Table 2: Compositions of gelatine-based films (5% w/V)

Table 3: Compositions of cellulose-based films (2 and 5% w/V)

Table 4: Compositions of alginate-based films (1% w/V)

Table 5: Compositions of Hyaluronate-based films (1% w/V)

Table 6: Compositions of films based on mixed polymers

Procedures for the preparation of exemplary films

General procedures

As observable from the following preparation protocols, chitosan-based films were prepared at a concentration of 1% and 2% w/V chitosan with respect to the total volume of the solution (i.e. 200 mg chitosan or 400 mg chitosan in 20 ml solution, respectively). When slime is present, the 200 mg of chitosan (in case of film with 1% w/V chitosan) are solubilized in a volume of acidified HPW lower than the 20 ml of the total solution volume according to the V/V ratio of added slime as described in Table 1. For example, in CS_7030 films, 6 ml are slime and 14 ml are chitosan in 1% w/V acid solution, while in CS_3070 films the millilitres of slime are 14 and those of chitosan in 1% w/V acid solution are 6, as indicated in Table 1.

Three different acids (HC1, Acetic Acid, and Lactic Acid) were used for chitosan solubilization, and the possibility of dissolving chitosan directly into pure snail slime was also evaluated. Regarding the use of 6M HC1 it has been observed that the solubilisation of chitosan occurs fairly quickly as opposed to when using Acetic Acid or Lactic Acid in which the solution was heated to 37 °C or 30 °C for two hours. In the case of chitosan 2% w/V, Acetic Acid, and Lactic Acid were used for chitosan solubilisation. Direct solubilisation in pure snail slime involved times comparable to those obtained using HC1. The solubilisation of chitosan requires different times depending on the amount of acid water that is added: the higher the initial concentration, the longer the time required for solubilisation. For the preparation of gelatine-based films, gelatines of different origins may be used. In the following Examples, a gelatine extracted from porcine skins (Sigma, 300 Bloom) and one of bovine origin (Sigma, 225 Bloom) are used. The preparation method followed for both is the same, therefore a unique description is given in the same paragraph.

The exemplary gelatine-based films were prepared at a concentration of 5% w/V gelatine with respect to the total volume of the solution (i.e. 1 g of gelatine in 20 ml solution). It should be understood that other concentrations of gelatine, in particular from 0.5 to 40% w/V, could be used. When slime is present, the 1 g of gelatine is solubilized in a volume of HPW lower than the 20 ml of total solution volume according to the V/V ratio of added slime as described in Table 2.

For the preparation of the cellulose-based films, hydroxypropyl methylcellulose HPMC E5 and E50 (Dow Chemical Company, USA) and sodium carboxymethyl cellulose (CMC, ACEF Piacenza, Italy) were used, but other types of cellulose could also be used. The exemplary cellulose-based films were prepared at a concentration equal to 5% w/V of E5 and E50 (i.e. 1 g of E5 or E50 in 20 ml of solution) and equal to 2% w/V of CMC (i.e. 400 mg of CMC in 20 ml of solution) with respect to the total volume of the solution. Other concentrations of cellulose could also be used, for example from 0.1 to 15% w/V for E5 and from 0.5 to 3% w/V for CMC. When S is present, 1 g of E5, 1 g of E50 or 400 mg of CMC is solubilized in a volume of HPW lower than 20 ml or equal to the total volume of the solution according to the V/V ratio of added snail slime, as described in Table 3. For the S_100 films, 1 g of E5, 1 g of E50 or 400 mg of CMC is solubilized in 20 ml of snail slime.

For the preparation of the alginate-based films, sodium alginate (Fluka) was used at a concentration of 1% w/V, with respect to the total volume of the solution (i.e. 150 mg of AL in 15 ml of solution). Other concentrations of alginate could also be used, for example from 0.5 to 3% w/V. When S is present, 150 mg of AL are solubilized in a volume of HPW lower than 15 ml, according to the V/V ratio of added snail slime, as described in Table 4. For the preparation of the hyaluronate-based films, sodium hyaluronate (ACEF spa, Italy) was used at a concentration of 1% w/V, with respect to the total volume of the solution (i.e. 150 mg of H in 15 ml of solution). Other concentrations of hyaluronate could also be used, for example from 0.1 to 2% w/V. When S is present, 150 mg of H are solubilized in a volume of HPW lower than 15 ml, according to the V/V ratio of added snail slime, as described in Table 5. For the S 100 films, 150 mg of H are solubilized in 15 ml of snail slime.

For the preparation of mixed polymers-based films, sodium carboxymethyl cellulose (CMC, ACEF Piacenza, Italy) was used at a fixed concentration of 2% w/V, while Hyaluronate (ACEF spa, Italy) concentration was varied between 0.3-2% w/V, with respect to the total volume of the solution (i.e. 400 mg of CMC in 20 ml of solution). Other concentrations of cellulose could also be used, for example from 0.5 to 3% w/V. In all the films described in Table 6, the solid polymers (CMC and H) are directly solubilised in snail slime.

The following materials were employed:

• chitosan FG90 (100 kDa Faravelli, Italy);

• sodim hyaluronate (1650 kDa, ACEF spa, Italy);

• swine gelatine (Sigma, 300 Bloom);

• bovine gelatine (Sigma, 225 Bloom);

• citric acid (Merck, Germania);

• glycerol (Farmalabor, (MI), Italy);

• cellulose as HPMC, Methocel E5 and E50 (Dow Chemical Company, USA);

• cellulose as sodium CMC (medium viscosity, ACEF, Piacenza, Italy);

• sodium alginate (Fluka);

• acetic acid and lactic acid (Sigma Aldrich).

Snail slime from Helix Aspersa Muller (kindly offered by“I Poderi” farm, Montemerano, Italy), extracted by MullerOne method (as described in http://www.mullerone.com/it/en/extraction-process), was used for the film’s preparation and stored at 4°C in a sealed polyethylene bottle until use. Moreover, other film’s composition were obtained by using the HelixComplex snail slime, extracted by HelixPharma srl (patent application publications N. IT 10207000117547 and N. WO 2013011371 Al). This extract was stored in the freezer at -20 °C and thawed immediately before use.

The following procedures were performed at room temperature, unless otherwise indicated. According to the Examples below, different amounts of slime and polymer solution are poured into a PE petri dish with diameters ranging from 5.5 to 8.7 cm and the film is obtained by solvent evaporation, as further detailed below. It is to be understood that the volume of such solution and the diameter of such petri dish indicated in the examples are merely exemplary and may be varied. In particular, by varying the volume of such solution and/or the diameter of the petri dish it is possible to obtain films of different thicknesses.

All the produced samples were stored at room temperature between two sheets of plastic- coated aluminium closed inside PVC bags.

Example 1: Procedure for the preparation of non-cross-linked chitosan-based films (1% w/V) by acidification with 6M HCl

C_HC1 film: Chitosan-based films were prepared by dissolving 200 mg of chitosan FG90 (100 kDa Faravelli, Italy) (1% w/V) in 20 mL of high purified water acidified with 6M HCl (0.75% v/v). 7.4 mL of this solution were poured into a PE (polyethylene) petri dish (5.5 cm diameter): the film is obtained by solvent evaporation (solvent casting) under a laminar hood at room temperature overnight.

CS_7030_HC1 films: The CS_7030_HC1 films were prepared by dissolving 200 mg of chitosan in 14 ml of high purified water acidified with 6M HCl (the volume of HCl is kept constant as in C HCl). Subsequently, after complete solubilisation of chitosan, which took place by stirring, 6 ml of (pure) slime were added and the mixture was allowed to stir for further 10 minutes. Finally, 7.4 ml of this mixture were poured into a 5.5 cm diameter PE petri dish. The film is obtained by evaporating the solvent under laminar hood at room temperature overnight.

CS_3070_HC1 films: The CS_3070_HC1 films were prepared by dissolving 200 mg of chitosan in 6 ml of high purified water acidified with 6M HCl (the volume of HCl is kept constant as in C HCl). Subsequently, after complete solubilisation of chitosan, which took place by stirring, 14 ml of (pure) slime were added and the mixture was allowed to stir for further 10 minutes. Finally, 7.4 ml of this mixture were poured into a 5.5 cm diameter PE petri dish. The film is obtained by evaporating the solvent under laminar hood at room temperature overnight.

The obtained films are reported in Table 1.

Example 2: Procedure for the preparation of non-cross-linked chitosan-based films (1% w/V) by acidification with 1% v/v acetic acid

C_A film: Chitosan-based films were prepared by dissolving 200 mg of chitosan (1% w/V) in 20 mL of an aqueous solution of 1% v/v acetic acid. The solution was maintained under stirring for two hours at 30 °C until complete dissolution of the chitosan. Finally, 7.4 mL of solution were poured into a PE petri dish (5.5 cm diameter) and allowed to evaporate under a laminar hood at room temperature overnight to obtain the film.

CS_8515_A film: The CS_8515_A films were prepared by dissolving 200 mg of chitosan in 17 ml of 1% v/v acetic acid acidified aqueous solution. The solution was maintained under stirring for two hours at 30 °C until complete dissolution of the chitosan. 3 ml of slime were added to the solution and the mixture was kept under stirring for further 10 minutes. Finally, 7.4 mL of mixture were poured into a PE petri dish (5.5 cm diameter): the film was obtained after evaporation of the solvent under laminar hood at room temperature overnight.

CS_7030_A film: The CS_7030_A films were prepared by dissolving 200 mg of chitosan in 14 mL of 1% v/v acetic acid acidified aqueous solution. The solution was maintained under stirring for two hours at 30 °C until complete dissolution of the chitosan. 6 ml of slime were added to the solution and the mixture was kept under stirring for further 10 minutes. Finally, 7.4 mL of mixture were poured into a PE petri dish (5.5 cm diameter): the film was obtained after evaporation of the solvent under laminar hood at room temperature overnight.

CS_3070_A film: The CS_3070_A films were prepared by dissolving 200 mg of chitosan in 6 mL of aqueous solution acidified with 1% v/v acetic acid. The solution was maintained under stirring for two hours at 30 °C until complete dissolution of the chitosan. 14 ml of slime were added to the solution and the mixture was kept under stirring for further 10 minutes. Finally, 7.4 mL of mixture were poured into a PE petri dish (5.5 cm diameter): the film was obtained after evaporation of the solvent under laminar hood at room temperature overnight.

The obtained films are reported in Table 1.

Example 3: Procedure for the preparation of non-cross-linked chitosan-based films (1% w/V) by acidification with 1% v/v lactic acid

C_L film: Chitosan-based films have been prepared by dissolving 200 mg of chitosan (1% w/V) in 20 mL of an aqueous solution of 1% v/v lactic acid. The solution was maintained under stirring for two hours at 37 °C until complete dissolution of the chitosan. Finally, 7.4 mL of solution were poured into a PE petri dish (5.5 cm diameter) and allowed to evaporate under a laminar hood at room temperature overnight to obtain the film. CS_7030_L film: The CS_7030_L films were prepared by dissolving 200 mg of chitosan in 14 mL of aqueous solution acidified with 1% v/v lactic acid. The solution was maintained under stirring for two hours at 37 °C until complete dissolution of the chitosan. 6 ml of slime was added to the solution and the mixture was kept stirring for further 10 minutes. Finally, 7.4 mL of mixture were poured into a PE petri dish (5.5 cm diameter): the film was obtained after evaporation of the solvent under laminar hood at room temperature overnight.

CS_3070_L film: The CS_3070_L films were prepared by dissolving 200 mg of chitosan in 6 mL of aqueous solution acidified with 1% v/v acetic acid. The solution was maintained under stirring for two hours at 37 °C until complete dissolution of the chitosan. 14 ml of slime was added to the solution and the mixture was kept under stirring for further 10 minutes. Finally, 7.4 mL of mixture were poured into a PE petri dish (5.5 cm diameter): the film was obtained after evaporation of the solvent under laminar hood at room temperature overnight.

The obtained films are reported in Table 1.

Example 4: Procedure for the preparation of non-cross-linked chitosan-based films (1% w/V) by direct solubilisation in snail slime

The employed snail slime is acidic, thus suitable for solubilization of chitosan, which needs pH values below 6 to dissolve. It follows that the use of S allows direct solubilization of chitosan through a‘green’ procedure and provides materials where the good characteristics of chitosan are enriched by the peculiar properties of snail slime. CS_7030_SOL film: The CS_7030_SOL films were prepared by dissolving 200 mg of chitosan in 6 mL of snail slime; the mixture was maintained under stirring for 60 minutes at room temperature; then 14 ml of high purified water was added and the mixture was kept under stirring for further 10 minutes. Finally, 7.4 mL of mixture were poured into a PE petri dish (5.5 cm diameter): the film was obtained after evaporation of the solvent under laminar hood at room temperature overnight.

CS_3070_SOL film: The CS_3070_SOL films were prepared by dissolving 200 mg of chitosan in 14 mL of snail slime; the solution was maintained under stirring for 60 minutes at room temperature; then 6 ml of high purified water was added and the mixture was kept under stirring for further 10 minutes. Finally, 7.4 mL of mixture were poured into a PE petri dish (5.5 cm diameter): the film was obtained after evaporation of the solvent under laminar hood at room temperature overnight. The obtained films are reported in Table 1.

Example 5: Procedure for the preparation of chitosan-based film cross-linked with 0.1% w/V hyaluronate

This Example refers to the preparation of cross-linked chitosan films, prepared by acidification with acetic acid or by direct solubilisation in snail slime and subsequent diffusion. Crosslinking was performed by diffusion of a solution of sodium hyaluronate (“( 'hitosan hyaluronic acid hybrid film as a novel wound dressing: in vitro and in vivo studies’’ Polym. Adv. Technol. 2007; 18: 869-875 and“ Alginate membranes loaded with hyaluronic acid and silver nanoparticles to foster tissue healing and to control bacterial contamination of non-healing wounds’’ Journal of Materials Science: Materials in Medicine 2018; 29: 22) on 1% w/V chitosan films previously prepared as described in Examples 2 and 4, employing a method reported in literature for the preparation of scaffolds with lower amounts of hyaluronate and then suitably modified for the preparation of films, as described below.

The advantage of cross-linking chitosan with hyaluronate resides in that hyaluronate is one of the fundamental components of the extracellular matrix of connective tissue. It has also been studied in several biomedical applications for its role in wound healing procedures (stimulates cell proliferation and migration).

C_A+ H film: Chitosan films were initially prepared as described above for the sample C_A in Example 2. The following day 7.4 ml of a 0.1% w/V aqueous solution of sodium hyaluronate (1650 kDa, ACEF spa, Italy) were deposited on the film kept inside the petri dish. The hyaluronate solution gradually diffuses into the chitosan film: the diffusion procedure is completed in about 1 hour, after which the material is left to dry under a laminar hood overnight.

CS_7030_A+ H film: Chitosan films were initially prepared as described above for sample CS_7030_A in Example 2. The following day 7.4 ml of a 0.1% w/V aqueous solution of hyaluronate were deposited on the film kept inside the petri dish. After diffusion, which occurs as described above, of the hyaluronate solution within the film, the material is left to dry under a laminar hood overnight.

CS_3070_A+ H film: Chitosan films were initially prepared as described above for the sample CS_3070_A in Example 2. The following day 7.4 ml of a 0.1% w/V aqueous solution of hyaluronate were deposited on the film kept inside the petri dish. After diffusion of the hyaluronate solution within the film, which occurs as described above, the material is left to dry under a laminar hood overnight.

CS_3070_SOL+ H film: Chitosan films were initially prepared as described above for the sample CS_3070_SOL in Example 4. The following day 7.4 ml of a 0.1% w/V aqueous solution of hyaluronate were deposited on the film kept inside the petri dish. After diffusion, which occurs as described above, of the hyaluronate solution within the film, the material is left to dry under a laminar hood overnight.

The obtained films are reported in Table 1.

Example 6: Procedure for the preparation of non-cross-linked films based on chitosan (2% w/V)

C2_A Gly film: The C2_A Gly films were prepared by dissolving 400 mg of chitosan (2% w/V) in 20 mL of an aqueous solution of 2% v/v acetic acid. The solution was maintained under stirring for two hours at 37 °C until complete dissolution of the chitosan. Subsequently, 30% w/w glycerol was added (with respect to chitosan weight) and the solution was maintained under stirring for further ten minutes. Finally, 7.4 mL of solution were poured into a PE petri dish (5.5 cm diameter) and allowed to evaporate under a laminar hood at room temperature overnight to obtain the film.

C2S_3070_A Gly film: The C2S_3070_A Gly films were prepared by dissolving 400 mg of chitosan (2% w/V) in 6 mL of a 2% v/v aqueous acetic acid solution and the solution was kept stirring for two hours at 37 °C until complete dissolution of the chitosan. Subsequently, 14 ml of snail slime and 30% w/w glycerol (with respect to chitosan weight) were added. The mixture was kept under stirring for ten minutes at 37°C, then 7.4 mL of solution were poured into a PE petri dish (5.5 cm diameter) and allowed to evaporate under a laminar hood at room temperature overnight to obtain the film.

C2S_7030_A Gly film: The C2S_7030_A Gly films were prepared by dissolving 400 mg of chitosan (2% w/V) in 14 mL of a 2% v/v aqueous acetic acid solution and the solution was kept stirring for two hours at 37 °C until complete dissolution of the chitosan. Subsequently, 6 ml of snail slime and 30% w/w glycerol (with respect to chitosan weight) were added. The mixture was kept under stirring for ten minutes at 37°C, then 7.4 mL of solution were poured into a PE petri dish (5.5 cm diameter) and allowed to evaporate under a laminar hood at room temperature overnight to obtain the film.

C2S_7030_A film: C2S_7030_A films were prepared by dissolving 400 mg of chitosan in 14 mL of aqueous solution acidified with 2% v/v of acetic acid. The solution was maintained under stirring for two hours at 37 °C until complete dissolution of the chitosan. 6 ml of slime was added to the solution and the mixture was kept stirring for further 10 minutes. Finally, 7.4 mL of mixture were poured into a PE petri dish (5.5 cm diameter): the film was obtained after evaporation of the solvent under laminar hood at room temperature overnight.

C2_L Gly film: The C2_L Gly films were prepared by dissolving 400 mg of chitosan (2% w/V) in 20 mL of an aqueous solution of 2% v/v lactic acid. Subsequently, 30% w/w glycerol was added (with respect to chitosan weight). The solution was maintained under stirring for two hours at 37 °C until complete dissolution of the chitosan. Finally, 7.4 mL of solution were poured into a PE petri dish (5.5 cm diameter) and allowed to evaporate under a laminar hood at room temperature overnight to obtain the film.

C2S_3070_L Gly film: The C2S_3070_L Gly films were prepared by dissolving 400 mg of chitosan (2% w/V) in 6 mL of a 2% v/v aqueous lactic acid solution and the solution was kept under stirring at 37 °C for two hours until complete dissolution of the chitosan. Subsequently, 14 ml of snail slime and 30% w/w glycerol (with respect to chitosan weight) were added. The mixture was left under stirring for ten minutes and finally, 7.4 mL of solution were poured into a PE petri dish (5.5 cm diameter) and allowed to evaporate under a laminar hood at room temperature overnight to obtain the film.

C2S_7030_L Gly film: The C2S_7030_L Gly films were prepared by dissolving 400 mg of chitosan (2% w/V) in 14 mL of a 2% v/v aqueous lactic acid solution and the solution was kept under stirring at 37 °C for two hours until complete dissolution of the chitosan. Subsequently, 6 ml of snail slime and the 30% w/w glycerol (with respect to chitosan weight) were added. The mixture was left under stirring for ten minutes and finally, 7.4 mL of solution were poured into a PE petri dish (5.5 cm diameter) and allowed to evaporate under a laminar hood at room temperature overnight to obtain the film.

The obtained films are reported in Table 1.

Example 7: Procedure for the preparation of non-cross-linked films based on gelatine GP and GB films: Gelatine-based films were prepared by placing 1 g of gelatine in 20 mL of high purified water (5% w/V). The solution was maintained under stirring at 38 °C for 30 minutes until the gelatine was completely solubilized. 7.4 mL of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight. Films obtained from swine gelatine are indicated below by the acronym GP, while those obtained from bovine gelatine by the acronym GB.

GPS_7030 and GBS_7030 films: The GPS 7030 and GBS 7030 films were prepared by placing 1 g of gelatine in 14 ml of high purified water. The solution was maintained under stirring at 38 °C for 30 min until the gelatine was completely solubilized, then 6 ml of slime (pure) were added and the mixture was kept under stirring for 5 min without heating. 7.4 mL of this mixture were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight.

GPS_3070 and GBS_3070 film: The GPS 3070, GBS 3070 films were prepared by placing 1 g of gelatine in 6 ml of high purified water. The solution was maintained under stirring at 38 °C for 30 min until the gelatine was completely solubilized: then 14 ml of slime (pure) were added and the mixture was kept under stirring for 5 min without heating. 7.4 mL of this mixture was poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight.

The obtained films are reported in Table 2.

Example 8: Procedure for the preparation of gelatine-based films cross-linked with citric acid 30% (w/w) with respect to gelatine and addition of glycerol

Crosslinking of gelatine was carried out using citric acid (“ Citric acid-incorporated fish gelatine/chitosan composite films’’ Food Hydrocolloids 2019; 86: 95-103). Among the different methods reported in literature for cross-linking gelatine it was decided to use citric acid, because it has numerous advantages. Citric acid is an additive widely used in both food and medicinal products (e.g. effervescent forms). Furthermore, films cross- linked with citric acid maintain the characteristic colour and transparency typical of gelatine (as opposed to materials cross-linked with glurtaraldehyde, or genipine, which become orange and dark blue respectively).

Citric acid was used for cross-linking in an amount of 30% (w/w) with respect to the gelatine. Glycerol was added to the films of the invention as a humectant in order to keep the amount of residual water present in the film constant during storage.

GP_Gly_CA and GB_Gly_CA films: Films were prepared by placing 1 g of gelatine and 300 mg of citric acid (Citric acid, Merck, Germany) in 20 mL of high purified water. The solution was maintained under stirring at the temperature of 38 °C until complete solubilization of the two components, then 30 microlitres of 5M NaOH were added to obtain a pH value of about 7. The mixture was heated to 60 °C and kept at this temperature for 30 minutes, then 300 mg of glycerol were added and the solution kept under stirring for five minutes. 7.4 mL of this mixture were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight. Snail slime can be inserted into these films.

GPS_Gly_4060 CA and GBS_Gly_4060 CA films: The GPS_Gly_4060 CA e GBS_Gly_4060_CA films were prepared by placing 1 g of gelatine (either from porcine or bovine origin) and 300 mg of citric acid (Citric acid, Merck, Germany) in 8 mL of high purified water. The solution was maintained under stirring at the temperature of 38 °C until complete solubilization of its two components, then 30 microlitres of 5M NaOH have been added to obtain a pH value of about 7. The mixture was heated to 60 °C and maintained at this temperature for 30 minutes. After such time the mixture was cooled to 37°C under stirring and 12 ml of slime and 300 mg of glycerol were added. The mixture was kept under stirring for 5 minutes, then 7.4 mL of this mixture were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight.

GPS Gly _ 5050 CA and GBS_Gly_5050 CA films: GPS Gly _ 5050 CA and

GBS_Gly_5050_CA films were prepared by placing 1 g of gelatine and 300 mg of citric acid (Citric acid, Merck, Germany) in 10 mL of high purified water. The solution was kept stirring at the temperature of 38 °C until complete solubilization of its two components, then 30 microlitres of 5M NaOH have been added to obtain a pH value of about 10. The mixture was heated to 60 °C and maintained at this temperature for 30 minutes. After this time the mixture is cooled to 37 °C under stirring after which 10 ml of slime and 300 milligrams of glycerol were added. This amount corresponds to a glycerol mass of 30% (w/w with respect to the amount of gelatine). The mixture was kept under stirring for 5 minutes, then 7.4 mL of this mixture were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight.

The obtained films are reported in Table 2.

Example 9: Procedure for the preparation of non-cross-linked hydroxy propyl methyl cellulose-based films 5% w/V Preparation of E5 and E50-based films: E5 and E50 films were obtained by dissolving 1 g of E5 or E50 in 20 mL of HPW (5% w/V) under gentle stirring overnight to avoid bubble formation. Then, 10.2 g of this solution were poured in a polyethylene Petri dish (0= 8.5 cm) and allowed to dry under a laminar hood at room temperature overnight. The obtained films were labeled E5 or E50 and stored at room temperature between two sheets of plastic-coated aluminum closed inside PVC bags.

Preparation of E5 and E50-based films containing snail slime:

E5S 7030 and E50S_7030 films: The E5S_7030 and E50S_7030 films were prepared by placing 1 g of cellulose in 14 ml of highly purified water. The solution was kept under stirring at room temperature overnight until complete solubilization of the cellulose, then 6 ml of snail slime were added and the mixture was kept under stirring for further 5 minutes. 10.2 grams of this mixture were poured into a PE petri dish (8.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight.

E5S 3070 and E50S_3070 films: The E5S_3070 and E50S_3070 films were prepared by placing 1 g of cellulose in 6 ml of high purified water. The solution was kept under stirring at room temperature overnight until complete solubilization of the cellulose, then 14 ml of snail slime were added and the mixture was kept under stirring for further 5 minutes. 10.2 grams of this mixture were poured into a PE petri dish (8.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight.

E5S_100 and E50S_100 films: E5S_100 and E50S_100 films were prepared by placing 1 g of cellulose in 20 ml of of snail slime under gentle stirring overnight. Then 10,2 grams of this solution were poured in petri dish (0= 8.5 cm) and allowed to dry under a laminar hood at room temperature overnight.

The obtained films were labeled as reported in Table 3.

Example 10: Preparation procedure of films containing sodium carboxymethyl cellulose 2% (w/V)

Preparation of CMC-based films: CMC -based films were obtained by dissolving 0.4 g of CMC in 20 mL of HPW (2% w/v) under gentle stirring overnight. Then, 10.2 g of this solution were poured in a polyethylene Petri dishes (0= 8.5 cm) and allowed to dry under a laminar hood at room temperature overnight. Preparation of CMC-based films containing snail slime: The CMCS 3070 films were prepared by dissolving 0.4 g of CMC into 6 ml of HPW under gentle stirring overnight. Then 14 ml of snail slime were added and the solution maintained under stirring for at least 30 minutes until no bubbles were present. 10,2 grams of this solution were poured in petri dish (0= 8.5 cm) and allowed to dry under a laminar hood at room temperature overnight. The CMCS_100 films were prepared by dissolving 0.4 g of CMC into 20 ml of snail slime under gentle stirring overnight. Then 10,2 grams of this solution were poured in petri dish (0= 8.5 cm) and allowed to dry under a laminar hood at room temperature overnight.

The obtained films were labeled as reported in Table 3.

Example 11: Preparation procedure of sodium alginate-based films 1% (w/V)

Alginate (AL) films with different percentages in volume of snail slime were produced (Table 4). In particular, 15, 30 and 50% V/V of S were involved in the preparation of the composite films, adding the relative volume of S to the 1 % w/V solution of AL dissolved at RT in the remaining volume of water.

AL films: AL films were prepared by placing 0.2 g of sodium alginate in 20 mL of HPW (1% w/V). The solution was kept under stirring at room temperature overnight until complete solubilization of the alginate. 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature.

ALS_8515 films: ALS_8515 films were prepared by placing 0.2g of sodium alginate in 17 ml of HPW. The solution was kept under stirring at room temperature overnight until complete solubilization of the polymer, then 3 ml of snail slime were added and the solution was kept under stirring for 30 minutes at room temperature. 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature

ALS_7030 films: ALS_7030 films were prepared by placing 0.2g of sodium alginate in 14 ml of HPW. The solution was kept under stirring at room temperature overnight until complete solubilization of the polymer, then 6 ml of snail slime were added and the solution was kept under stirring for 30 minutes at room temperature. 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature

ALS_5050 films: ALS_5050 films were prepared by placing 0.2g of sodium alginate in 10 ml of HPW. The solution was kept under stirring at room temperature overnight until complete solubilization of the polymer, then 10 ml of snail slime were added and the solution was kept under stirring for 30 minutes at room temperature. 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature.

The obtained films were labeled as reported in Table 4.

Example 12: Preparation procedure of sodium hyaluronate (H)-based films 1% (w/V)

H films: H films were prepared by placing 0.2 g of Hyaluronate in 20 mL of HPW (1% w/V). The solution was kept under stirring at room temperature overnight until complete solubilization of the hyaluronate. 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature.

HS_7030 films: HS_7030 films were prepared by placing 0.2g of hyaluronate in 14 ml of HPW. The solution was kept under stirring at room temperature overnight until complete solubilization of the hyaluronate, then 6 ml of snail slime were added and the solution was kept under stirring for 30 minutes at room temperature. 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature.

HS_5050 films: HS_5050 films were prepared by placing 0.2g of hyaluronate in 10 ml of HPW. The solution was kept under stirring at room temperature overnight until complete solubilization of the hyaluronate, then 10 ml of snail slime were added and the solution was kept under stirring for 30 minutes at room temperature. 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature.

HS_100 films: HS_100 films were prepared by placing 0.2g of hyaluronate in 20 ml of snail slime. The solution was kept under stirring at room temperature overnight until complete solubilization of the hyaluronate, then 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature.

HS_7030 Gly films: HS_7030 Gly films were prepared by placing 0.2g of hyaluronate in 14 ml of HPW. The solution was kept under stirring at room temperature overnight until complete solubilization of the hyaluronate, then 6 ml of snail slime and 30 mg of glycerol were added and the solution was kept under stirring for 30 minutes at room temperature. 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room.

HS_100 Gly films: HS_100 Gly films were prepared by placing 0.2g of hyaluronate in 20 ml of snail slime. The solution was kept under stirring at room temperature overnight until complete solubilization of the hyaluronate, then 30 mg of glycerol were added and the solution was kept under stirring for 30 minutes at room temperature. 7.4 ml of this solution were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature.

The obtained films were labeled as reported in Table 5.

Example 13: Preparation procedure of films based on carboxymethyl cellulose and sodium hyaluronate

CMC_H0.3_S100Gly films: CMC_H0.3_S100Gly films were prepared by dissolving 0,4 g of CMC into 20 ml of snail slime at room temperature overnight. Then 60 mg of hyaluronate and 60 mg of glycerol were added and the solution stirred at room temperature for about 30 minutes. Then, 10.2 g of this solution were poured in each Petri dish (0= 8.5 cm) and put under laminar flow hood overnight.

CMC_Hl_S100Gly Films: CMC_Hl_S100Gly films were prepared by dissolving 0,4 g of CMC into 20 ml of snail slime at room temperature overnight. Then 0,2 g of hyaluronate and 60 mg of glycerol were added and the solution stirred at room temperature for about 30 minutes. Then, 10.2 g of this solution were poured in each Petri dish (0= 8.5 cm) and put under laminar flow hood overnight.

CMC_H2_S100Gly Films: CMC_H2_S100Gly films were prepared by dissolving 0,4 g of CMC into 20 ml of snail slime at room temperature overnight. Then 0,4 g of hyaluronate and 60 mg of glycerol were added and the solution stirred at room temperature for about 30 minutes. Then, 10.2 g of this solution were poured in each Petri dish (0= 8.5 cm) and put under laminar flow hood overnight.

CMC_Hl_S100Glyl0 Films: CMC_Hl_S100Glyl0 films were prepared by dissolving 0,4 g of CMC into 20 ml of snail slime at room temperature overnight. Then 0,2 g of hyaluronate and 2 ml of glycerol were added and the solution stirred at room temperature for about 30 minutes. Then, 10.2 g of this solution were poured in each Petri dish (0= 8.5 cm) and put under laminar flow hood overnight.

CMC_H2_S100Glyl0 Films: CMC_H2_S100Glyl0 films were prepared by dissolving 0,4 g of CMC into 20 ml of snail slime at room temperature overnight. Then 0,4 g of hyaluronate and 2 ml of glycerol were added and the solution stirred at room temperature for about 30 minutes. Then, 10.2 g of this solution were poured in each Petri dish (0= 8.5 cm) and put under laminar flow hood overnight.

The obtained films are reported in Table 6.

Characterization of films

Example 14: Mechanical properties

The mechanical properties of the films are useful for assessing their suitability for topical application (e.g. cutaneous and mucosal). For the mechanical characterisation, uniaxial tensile tests were carried out, fixing the rectangular specimen to two clamps and recording its mechanical behaviour obtaining stress/strain graphs. In particular, the variation of the following parameters was evaluated: stress at break (<_¾), elongation at break (ei,), Young modulus (E) and maximum stress (o_m). During a constant speed tensile test, the crossbar will move by the same amount over the time interval. The resistance that the material opposes to the displacement of the crossbar is overcome by applying progressively greater loads. The higher the load to be applied, the greater the tensile strength of the material.

The stress is obtained by dividing the load (applied to the sample, point by point) by the area on which the load acts (such area is obtained by multiplying the width of the specimen by its thickness). It is preferred to use the stress magnitude rather than the load because stress represents a normalized value, and thus allows for a comparison of results from samples of different sizes. In this way, the maximum stress (highest value of the stress-strain curve) and stress at break, which corresponds to the stress recorded at the breaking time of the sample, were evaluated. Depending on the shape of the stress-strain curve (which depends on the characteristics of the tested material) stress at break and maximum stress may coincide or be different.

The strain (deformation) represents the elongation of the sample subjected to traction: again, the value is normalized in order to compare samples of different initial lengths. The strain is therefore expressed as the percentage of elongation of the sample with respect to its initial length. The elastic modulus (E) represents the tangent to the stress-strain curve in the section of elastic behaviour, then in the first section of the curve for strain values not exceeding 5%. It is a measure of sample stiffness: the higher E is, the greater the stress required to achieve the same strain.

The parameters examined provide information on the stiffness and fragility of the films. For cutaneous and/or mucosal and/or nail application, low stress at break (<_¾), Young modulus (E) and maximum stress (o_m) values and high elongation at break (e_b) are desirable. Indeed, these values indicate high deformability, extensibility, and reduced handling, stiffness and fragility.

The film thickness was measured by MITUTOYO digital micrometer with a range of 25 mm and a sensitivity of 0.001 mm at six different positions in each specimen. Using the volume of 7.4 mL, indicated in the exemplary preparation of the films, the mean thicknesses of 1% w/V chitosan-based films are about 50 pm, while those based on 5% w/V gelatine are about 120 pm. Following the addition of the (pure) snail slime, the average thicknesses increase, reaching values of about 130 pm for CS 3070 films and 180 pm for GS 3070 films, regardless of the type of gelatine used. The thickness of the film is a function of both the content of polymer used and the content of dry matter present in the snail slime (the dry matter is between 3% and 7% w/w, the rest is water). It is important to define the thickness of the film obtained, since other properties of the films depend on this characteristic, such as residual humidity, water vapour permeability and rate of dissolution and release of the active substances.

The samples were cut into strips 40 mm long and 4 mm wide, the ends of each strip were fixed with the clamps as shown in Figure 2 and the measurement was taken. Measurements were performed using an INSTRON 4465 dynamometer, connected and managed by a SERIES IX software for Windows. The load cell is 1 KN and the crossbar speed was set to 5 mm/min. Stress-strain curves were recorded at a crosshead speed of 5mm/min by the software SERIE IX for Windows. Six measurements were performed for each sample and the results are expressed as mean ± standard deviation and reported in Tables 7, 9 and 10. Mechanical tests were carried out on the samples one day after preparation.

Table 7: Mechanical properties of chitosan-based films

From the results of the mechanical properties in Table 7, it can be observed that as the slime content increases, the values of the modulus (E), e.g. from 1335 to 0.9 MPa for films prepared in acetic acid, and of the maximum stress (a_m) and stress at break (o_b) decrease significantly while the elongation at break (e_b) increases considerably, passing from values around 6% for films of chitosan only to over 100% for the film CS 3070 A. These values indicate an important change in the mechanical properties of the films due to the presence of slime: at high amounts, it greatly improves the extensibility of the films by reducing their rigidity. The materials thus obtained are therefore less fragile than chitosan alone and can be easily stretched by even 100% without breaking exerting a much lower force. This behaviour is very advantageous if the film is to be applied in areas subjected to continuous stresses, such as the groove of the arm or the palm of the hand, for example: in fact, the high deformability of the film and its reduced rigidity mean that the material can accommodate the movements without breaking. The prepared materials display both elastic and plastic properties.

As a general consideration, the flexibility of the films and their adhesiveness increase with S volume.

All the chitosan-based films appeared transparent, with colour gradually turning to yellow on increasing the amount of Snail slime (Fig. 3(A)). The SEM images of some chitosan films containing Snail slime are reported as example in Fig. 3(A): all the films revealed a smooth surface without uneven areas. SEM images were obtained with Philips XL-20 Scanning Electron Microscope. As reported in Table 8, films thickness is significantly affected by the acid used for chitosan dissolution: the thickness increase from acetic to lactic acid films can be ascribed to the increasing dimensions of the counterion [A. Begin, M.R. Van Calsteren, Antimicrobial films produced from chitosan, Int. J. Biol. Macromol. 26 (1) (1999) 63- 67.]. A significantly greater augmentation of the values of thickness occurs on increasing the S content, most likely as a consequence of the increasing amount of dry matter (dry matter content of S after lyophilization: 5%m/V). The influence of the nature and composition of the film forming solution on thickness is clearly shown by the results obtained for the C SOL samples, where water addition does not significantly affect the values of thickness [S. Khoshgozaran-Abras, M.H. Azizi, Z. Hamidy, N. Bagheripoor- Fallah, Mechanical, physicochemical and color properties of chitosan based-films as a function of Aloe vera gel incorporation, Carbohydr. Polym. 87 (2012) 2058-2062.].

Table 8: Thickness of chitosan-based films

Both C L and C_A films are rigid and brittle with high values of elastic modulus (E) and stress at break (<_¾), whereas C SOL films exhibit a relatively high extensibility (e_b) and low values of E and O_b. In agreement, S addition to C L and C_A compositions greatly influences the mechanical properties of the films, as clearly shown in Fig. 4(A): 8_b increases with S content while O_b and E decrease. The same trend is observed on going from CS 3070 SOL to C SOL, in agreement with the increase of S content. The effect produced by S addition is similar to that obtained by the introduction of plasticizers into the composition of chitosan films [C. Caner, et ah, J. Food Sci. 63 (6) (2006) 1049-1053; D. Lourdin, et ah, J. Appl. Polym. Sci. 63 (8) (1997) 1047-1053; L.F. Boesel, Carbohydr. Polym. 115 (2015) 356-363.]. The increased extensibility of the films at higher S concentration can be attributed to Snail slime-polymer interactions, which reduce the intermolecular interactions between polymer chains, facilitating their sliding and mobility and improving the overall extensibility.

Table 9: Mechanical properties of bovine and porcine gelatine films

From the data shown in Table 9, it can be seen that films obtained from gelatine-only, both bovine and porcine, are very rigid and fragile (high elastic moduli, low deformation percentage). Adding slime completely modifies the mechanical properties of the films that become extremely deformable and flexible as observable from Figure 3(B).

In Figure 4(B) are reported, by way of example, the stress-strain curves obtained on the samples GB and GBS 3070. The GB sample has a high stress at break (higher than 80 MPa), which also coincides with the maximum stress, low elongation capacity (maximum elongation of about 5%) and a high elastic modulus, as observable from the high slope of the first section of the curve, indicating a high stiffness. By contrast, the GBS 3070 sample has a curve characterised by high elongation (around 70%) corresponding to an extremely low stress at break value (less than 5 MPa). The curve has two sections: a first elastic section, up to deformation values of about 4-5%, followed by a plastic behaviour until breakage. This behaviour is often observed in polymers: the elastic section corresponds to the initial elastic deformation (the material can return to its original shape if the external stress is eliminated, that is, if tension is no longer exerted), while the plastic section is a consequence of the breakage of the interactions that hold the macromolecular chains together, which can thus easily slide on top of each other reaching high deformations at almost constant stresses. In this curve, maximum stress and stress at break do not coincide: the maximum stress is obtained at the end of the elastic section and is proportional to the load that must be applied to break the interchain interactions. The stress at break is measured at the time of breaking and may be lower than the maximum stress. The film has a very low elastic modulus (see slope of the line interpolating the first elastic section of the curve), and this indicates that the addition of slime makes the material much less rigid/stiff. Adding slime in such amount substantially changes the mechanical properties of the material, which from rigid becomes plastic.

Table 10: Mechanical properties of cellulose-based films

The stress at break decreases on increasing snail slime content, while the elongation at break increases for all the tested samples: however, the maximum extent of deformation is strongly influenced by the cellulose used (compare E50 and CMC, for example). CMC films are brittle, rigid (high elastic modulus) and stretch up to few units percent. After addition of snail slime, films become elastic (low elastic modulus) and their deformation at break reach values above 60%. A similar behaviour is observed for HPMC -based films, even they stretch up less than CMC after snail slime addition.

In Figure 4(C) are reported, by way of example, the stress-deformation curves obtained on the samples E5, E5S_7030 and E5S_3070. Sample E5 has a high stress at break (higher than 50 MPa), an elongation of just over 10% and a high elastic modulus, as can be seen from the high slope of the first section of the curve, indicating a high stiffness. Addition of slime in large quantities makes the material much less rigid and more easily stretchable. Films in the presence of slime are more deformable and flexible.

Figure 4(D) reports stress-strain curves obtained from CMC -based films. Films made of CMC are brittle and rigid since they have a high elastic modulus and break at few percent of deformation. Films become more stretchable as a consequence of snail slime addition: in particular, films CMCS 100 show an elastic behaviour and they break at about 60% of deformation.

From the comparison with the results obtained for the three different polymers, it is evident that the introduction of snail slime into the polymer solution greatly influences the mechanical properties of the film in all cases. As a matter of fact, all films prepared with the highest amount of slime stretch much more than the polymer alone: the elongation rates are about 20 times higher for chitosan-based films (see C_A vs CS_3070_A) and gelatine (see GB vs GBS 3070) and the maximum stress value (a_m) of gelatine-based films (GB vs GBS 3070) decreases by about 50 times while for chitosan-based films (C_A vs CS_3070_A) by about 20 times. This property makes the films extremely manageable (they can be subjected to different stresses without breaking) and allows them to be applied in a very wide range of treatments, such as on the skin, in points where they must adapt to surfaces of different shapes and in continuous movement.

Example 15: Adhesive and mucoadhesive properties

The films of the present invention were applied to human skin, nails and lip. Before applying the film, in some cases, the skin was slightly moistened with (drinking) water.

For example, in the case reported in Figure 5(A) two small drops of water have been dropped in the inner part of the hand palm which joins the thumb with the index finger. The film CS 3070 A was placed on the skin with a slight pressure for about ten seconds (time is a function of the film formulation). The film is perfectly anatomical and behaves like a second skin.

Leaving the film CS 3070 A adhered for 8 hours (from morning to afternoon), it can be observed in Figure 5(B) that the film is still perfectly anatomical even if subjected to continuous movements (typical hand movements); in all positions the film remains perfectly attached to the skin without ever detaching from it.

Also, in the groove of the arm, the film CS 3070 A remains perfectly adhered and is perfectly anatomical even during repeated arm movements, as shown in Figure 5(C). In this case, the film was left adhered for two hours. During this time it showed no signs of detachment.

Film CS 3070 A was also applied to a nail. Even in such case, the film perfectly adheres to the surface of the nail as it is observable form Figure 5(D), showing a particular of the nail itself.

Film CMC H1 S100 GlylO was applied to the lower lip previously moistened with two small drops of water. The film immediately adheres to the lip, as it is observable form Figure 5(E).

Film CMC H2 S100 GlylO was applies to the gingival mucosa. In this case the film immediately adheres to the mucosa without the need to wet the substrate, as it is observable form Figure 5(F).

Example 16: Tack test

The measurements were carried out on chitosan-based films using a rheometer (Anton Paar, modular compact rheometer MCR102) and/or by means of an INSTRON 4465 dynamometer. Work of adhesion and peak detachment force were used to evaluate the bioadhesive strength of the films. Figure 6 shows a detail of the moment of detachment of the CS 3070 A film from the pig skin during a measurement conducted by means of Instron 4465.

Pig skin (from Italian breeding) acquired from a butcher's shop, cut in disks of 2.5 cm diameter, were fixed to the base of the analytical support by acrylic glue (attack), after having been gently washed with detergent to remove fat and rinsed with phosphate buffer (pH = 7.4) and water repeatedly. The film was adhered to the pig rind using a fixed volume of phosphate buffer pH 7.4 (40 microL), left to adhere for 1 minute. Subsequently, the upper plunger, coated with double-sided tape (3M), was lowered until a force of 5 Newton (N) was applied to the film for 30 seconds. Subsequently, the plunger was raised at a speed of 1 mm/s, and the force required to detach the film from the pig rind was measured and expressed in Newton (N). A graph showing the force required to lift the piston at a constant speed on the y-axis and time (expressed in seconds) on the x-axis is obtained.

In Figure 6(B) curves obtained from CS 3070 L films during the tack tests measurements are reported as an example. The minimum of the curve represents the maximum adhesion force, the area under the curve represents the work that needs to be done to detach the film from the skin and is therefore a measure of the adhesion forces involved. The greater the area of the curve, the greater the capacity of the film to adhere to the skin. Data were collected by using the RheoCompass Software or the Instron Series IX package. Table 11 shows the curve area values obtained for some of the chitosan-based films prepared at 2% w/V.

Table 11 : Curve area values obtained for chitosan-based films prepared at 2% w/V

From the results it is observable that, depending on the acid used to dissolve chitosan, the films exhibit different adhesiveness. In particular, the film obtained by dissolving chitosan in lactic acid (C2_L Gly) has greater adhesion than the film solubilized in acetic acid (C2_A Gly), which is poorly adhesive. However, it is evident that the presence of the snail slime in the formulation leads to a significant increase in the adhesive capacity of the films: this value is around four times higher for the sample when used in the ratio polymenslime 30:70 v/v (see sample C2_A Gly vs. C2S_3070_A Gly).

The adhesive properties, expressed in terms of force needed for chitosan-based film detachment (F) and work of adhesion (W), are reported in Fig. 7. C_A films do not exhibit any adhesive performance. As a matter of fact, no F or W value could be measured due to the lack of adhesiveness of such film. However, the addition of S induces a certain adhesiveness, requiring a force up to about 10 N (CS_3070_A) to detach the films. A similar trend is observed for films prepared in lactic acid: C L exhibits an appreciable adhesive behaviour, which is further enhanced by S addition. As expected, the highest adhesive properties are recorded for films prepared by direct solubilization of chitosan into Snail slime. The increased flexibility and adhesion of S-containing films should improve the contact with skin, thus allowing a better penetration of film components into the tissue. Besides, the presence of polar groups into the snail slime, most probably belongings to glycolic acid, allantoin and proteins could increase the interactions.

The tack test was performed on cellulose films following the same procedure by fixing the film on aluminum plate and on a glass disk instead of pig’s skin. Cellulose-based films become sticky after S addition: the adhesive properties, expressed in terms of force needed for film detachment (F), are reported in Table 12. Table 12: Force (N) needed for film detachment from glass and aluminum substrates

Cellulose-based films exhibit good adhesive performances on both the substrates. Addition of snail slime enhances the adhesive properties of the films and the effect is more evident when the polymer is dissolved into the highest extract content.

Example 17: Evaluation of film solubility

Solubility evaluation is of particular relevance in order to understand the films stability in aqueous solutions and thus to identify a specific film application. In order to evaluate the solubility, the films were cut into squares of an area equal to 2.25 cm² and dried at 37 ° C, for a day. After that, they were weighted and immersed in 5 mL of HPW. After 24 hours the samples not completely dissolved were removed from water, and dried again at 37 ° C until a constant weight was obtained. Samples CMCS 100 and CMCS 3070 do not dissolve for more than two weeks while CS_8515_A, C2S_7030_A, C2S_7030_AGly do not dissolve for more than two months.

Considering the chitosan-based films (Table 1), most of the film composition containing the 1% w/V of the polymer dissolve within few minutes, thus acting as fast dissolving films. The only exception concerns the film composition with chitosan solubilized in 2% v/v acetic acid solution, at a C : S volume ratio of 85 : 15, which is insoluble in water for more than two months. As regards the films based on the 2% w/V of chitosan, only the composition with chitosan solubilized in 2% V/V acetic acid solution, at a C: S volume ratio of 70 : 30, and that containing also glycerol are insoluble in water for months. Therefore, a cross-linked film is obtained without using other chemical components. All the gelatin-based films (Table 2) are very soluble in water (dissolve in a time range from 20 to 240 minutes) as a function of the S content. In particular, films comprising swine gelatin are more soluble than films prepared with bovine gelatin.

HPMC -based films (Table 3) containing or not snail slime immediately solubilize when added to water, whatever their composition. A different and more interesting trend is found for CMC films: while pristine films solubilize in few minutes, compositions CMCS 3070 and CMCS 100 are able to preserve their structure for more than one week. In fact, after 24 hours the solubility was 45% and 60%, respectively, and these values remain approximately constant even after 7 days.

The extent of solubility was evaluated by the following equation:

weight of the sample after immersion

Solubility% =— . - . , _f - : - :— X 100

weight of the sample before immersion

The film composition based only on alginate (AL) quickly dissolved (Table 4) after few minutes while the film solubilization decreases increasing the snail slime content: ALS 7030 and ALS 5050 are water insoluble cross-linked films.

All the obtained hyaluronate based films (Table 5) and the CMC-hyaluronate mixed films (Table 6) swell in water and dissolved in few minutes, acting as fast dissolving films.

Example 18: Swelling degree

Approximately 30 mg of dry film were weighed for each of the gelatine-based film compositions examined, which were then immersed in 5 ml of phosphate buffer (PB) pH 7.4. After 1, 3, 5, 10, 20, 30, 60, 90, 120, 180, 240 minutes, each sample was extracted from the buffer, quickly dabbed on absorbent paper to remove surface water, and weighed to assess the progressive degree of swelling, i.e. to assess the amount of water absorbed by the sample. After being weighed, the sample is again immersed in the liquid. The degree of swelling of the films, indicates the capacity of the film to absorb water when placed in contact with the wet skin or in the aqueous environment that wets the mucous membranes and the extent of this swelling can modify the time necessary to promote adhesion, the mechanical properties of the films and finally the time of dissolution or release of a drug. The degree of swelling affects the drug release, so depending on the desired release rate, it is possible to modulate the swelling of the material.

In addition, measuring the degree of swelling allows having information on the degree of cross-linking of the material: as a matter of fact, the higher the cross-linking of a film, the lower the capacity of the film to absorb water. The swelling degree is therefore strongly influenced by the composition of the films, in particular by the type of hydrophilic polymer (presence of hydroxyl groups, amines and type of final chain), concentration of slime rich in mucopolysaccharides and other additives such as humectants and plasticisers.

The results, obtained by analysing the GB, GBS 3070, GP, GPS 3070 samples and shown below, are expressed as percentage of swelling as a function of time, according to the following formula:

Wet sample weight— Dry sample weight

Swelhng% = - - - - - - - x 100

Dry sample weight

Wherein: dry sample weight means the weight of the air-dried film before being immersed in PB and wet sample means the weight of the sample after each time of immersion in PB (times specified above).

With regard to gelatine-based films, the following results were obtained:

The GB films reach a swelling degree of 1000% after 4 h;

The films of GBS_3070 type reach the maximum swelling degree (500%) after 30 minutes, after which they begin to solubilise;

The GP films reach a swelling degree of 800% after 4 h;

The films of the GPS 3070 type also reach a swelling degree of 800% after 4 h.

The following conclusions can be drawn from the comparison of the above values.

Non-crossed-linked gelatine, both bovine and porcine, is a highly hydrophilic material, capable of absorbing large amounts of PB. At the same time, swine gelatine absorbs less water than bovine gelatine. The result should not be surprising, because the amount of water absorbed depends on several factors, including the Bloom index, which is a measure of the strength of the gel obtained under certain conditions and which in turn depends on the degree of renaturation that the protein can achieve when, after being solubilised in water, it is allowed to congeal. The degree of renaturation indicates the amount of formation of protein moieties in which the triple-helix structure is restored. Such moieties are stabilized predominantly by H bonds and interactions between polar groups: such interactions are the same as those that preside over the absorption of water molecules so that, due to the lack of polar groups available for absorption, the degree of swelling decreases. Since the Bloom index of the bovine gelatine is always lower than that of pig gelatine, the degree of renaturation of pig gelatine will be higher, resulting in a lower ability to absorb water. The GBS 3070 film is more soluble: in fact, after 30 minutes the sample weight tends to decrease, a clear indication that it is solubilizing.

The addition of slime to the GP film does not appear to affect the ability of the material to absorb water, nor does it affect its stability in an aqueous environment.

The explanation may lie in the different isoelectric point presented by the protein obtained from swine and from bovine: this involves interactions of different magnitudes with the slime, which, having a very acidic pH (2.5-3), certainly has positive charges.

Extremely fast solubilisation rates (less than 15 minutes for non-cross-linked materials) were observed for chitosan-based films.

The extent of dissolution of films of different composition allows to greatly expand the field of use thereof and to propose them for different applications: non-cross-linked chitosan-based films can be proposed for example for the topical treatment of cellulite imperfections. One of the advantages lies in the fact that the film, after being applied and having exerted its action, can be removed by simple cleaning with warm water. The product is completely natural and therefore does not pollute.

Insoluble films made of chitosan and snail slime (CS 8515 A, C2S 7030 A, C2S_7030_A Gly) show a degree of swelling which is about 150% for both the formulations not containing glycerol and 50% for the sample C2S_7030_A Gly after 24h of immersion in phosphate buffered solution.

Regarding cellulose-based films, the swelling behaviour was monitored for CMCS-3070 and CMCS 100, which are insoluble. Both the samples reach a degree of swelling of 100% after 24 hours, then the value does not change over time.

Example 19: Freeze drying of snail slime

The snail slime was freeze-dried as such and in addition to a lyoprotectant (dextran at 2 % w/w). The freeze-dried samples obtained, shown in Figure 8, preserve bioactivity, increase preservability over time and facilitate storage of the snail slime.

Example 20: Production and mechanical properties of films obtained with the freeze dried snail slime

Gelatine film using lyophilised snail slime (lyophilised in presence of 2 % w/w of dextran) were prepared with two different amounts of lyophilisate.

The GBS_3070_FDS films were prepared by placing 1 g of gelatine in 6 ml high purified water, the solution was maintained under stirring at 38 °C for 30 minutes until complete solubilization of the gelatine; the lyophilisate obtained from 14 mL of snail slime was re- suspended in the same volume of high purified water and finally added to the gelatine solution. The mixture was maintained under stirring for 5 min without heating. 7.4 mL of this mixture were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight.

The GBS_7030_FDS films were prepared by placing 1 g of gelatine in 14 ml of high purified water, the solution was kept under stirring at 38 °C for 30 minutes until complete solubilization of the gelatine; the lyophilisate obtained from 6 mL of snail slime was re suspended in the same volume of high purified water and finally added to the gelatine solution. The mixture was maintained under stirring for 5 min without heating. 7.4 mL of this mixture were poured into a PE petri dish (5.5 cm diameter): the film is obtained by solvent evaporation under a laminar hood at room temperature overnight.

The films thus obtained show a similar appearance to that obtained by using the pure (liquid) snail slime. Mechanical tests were conducted for the GBS 3070 FDS film. Stress at break c_¾ (MPa), elongation at break percentage 8_b (%), elastic modulus E (MPa) and maximum stress o_m (MPa) were measured according to the methods reported in Example 14. The obtained values are compared (Table 13) with the gelatine-only film.

Table 13. Measurement of stress at break c_¾ (MPa), elongation at break percentage 8_b (%), elastic modulus E (MPa), maximum stress o_m (MPa) for the film GBS 3070 FDS

Data shown in Table 13 confirm that the presence of the snail slime used in its lyophilised form provides the films plastic and highly extensibility properties, as obtained using the fresh (non-lyophilised) snail slime solution.

Example 21: Cell proliferation assay

Procedure 1: The C_A, CS 3070 A and CS 7030 SOL films were solubilized in 2 mL of sterile water then filtered (filters with 0.2 nm pores). The samples were then subjected to cell proliferation assay by CCK8 colorimetric test (Cell Counting Kit-8 Dojindo Molecular Technologies, Rockville, MD, EISA). Monkey kidney epithelial cells (VERO, ATCC CCL- 81) were used for the test. VERO cells, 24 hours prior to the experiment, were transferred to a 96-well flat bottom plate at the density of 10⁴ cells/100 pL of culture medium (Eagle's Essential Medium with addition of 1% Levoglutamine, 1% Streptopenicillin, 10% fetal bovine serum from Thermo Fisher Scientific US). The plate was incubated at 37 °C and 5% CO2. The exhausted medium was removed after 24 hours and the cellular monolayer washed with PBS (saline phosphate buffer). The cells were then incubated with 100 pL medium containing scalar dilutions (1 :2) of solubilized samples (starting at 1 : 10 dilution). After 72 hours of incubation at 37 °C, the exhausted medium was removed, the wells washed with PBS and finally fresh medium was added with 10 pL of CCK8 reagent. The plate was incubated at 37 °C for 2 hours. Finally, the plate was read at the spectrophotometer (OD450_nm) and the obtained value was compared with the positive control (cells grown in complete medium).

From the results shown in Figure 9, it is observable that the 3 films do not significantly interfere with proliferation (which is always higher than 70%).

Procedure 2: The effect of chitosan films on non-malignant epithelial cells metabolism was assessed in vitro after incubation of disks of C_A, CS_3070_A, CS_3070_SOL (0 = 6 mm) in 1 mL of Eagle's Minimal Essential Medium (MEM) at 37 °C for 24 h. Then, the media were used for the analysis on African green monkey kidney cells (Vero ATCC CCL-81). Briefly, cells were cultured in MEM supplemented with 10% fetal bovine serum (Carlo Erba Reagents, Milan, Italy), 100 U/mL penicillin and 100 pg/mL streptomycin at 37 °C with 5% CO2. For experiments, cells were seeded into 96-well plates at 10⁴ cells/well, and incubated at 37 °C for 24 h; subsequently, cell monolayer was washed with PBS and incubated with 100 pL of the different solutions, previously diluted twenty times in cell culture medium. Then, cell viability was assessed by aWST8-based assay according to the manufacturer's instructions (CCK-8, Cell Counting Kit-8, Dojindo Molecular Technologies, Rockville, MD, USA). After 72 h of incubation, cell monolayer was washed with PBS, and 100 pL of fresh medium containing 10 pL of CCK-8 solution were added. After 2 h at 37 °C, the absorbance was measured at 450/630nm; results were expressed as the percentage of absorbance relative to the untreated controls. Experiment was carried out in triplicate.

C L and C_A films did not interfere with Vero cells metabolism after 72 h of incubation (93.7% and 103.3%, respectively, and relative to untreated control cells) (Fig. 10). Addition of snail slime to these films induced an improvement in cell viability, especially for the samples of CA series. These samples exhibit a dose dependent increase in Vero viability as function of S content. The lowest cell viability was detected for C SOL films (72.7%); nevertheless, as a material is considered cytotoxic when its viability is <70% in comparison to untreated controls [UNI EN, ISO 10993-5, Biological Evaluation of MEDICAL DEVICES Part 5: Tests for in Vitro Cytotoxicity, 2009], all samples displayed a promising safety profile.

Example 22: Cytotoxicity and bioactivity evaluation

Human normal skin fibroblast BJ-5ta (ATCC, VA, USA, lot 63229591) were cultured in a 4: 1 (V/V) mixture of Dulbecco’s Modified Eagle’s Medium and Medium 199, supplemented with 10% FCS and 0.01 mg/ml hygromycin B. BJ-5ta cells were plated at a density of lxlO⁴ cells/cm² in 24-well plates containing the 2% w/V chitosan-based films (C2S_7030_A). Cells were also plated in wells for negative (CTR-, DMEM only) and positive (CTR+, DMEM + 0.05% phenol solution) controls. Plates were cultured in standard conditions, at 37 ± 0.5°C with 95% humidity and 5% ± 0.2 CO2 up to 72 hours. Cell viability was measured at 24 and 72 hours by Alamar Blue reagent (Cell Viability Reagent, LIFE Technologies Corp., Oregon, USA), added (1: 10 v/v) to each well and incubated for further 4 hours at 37°C. A redox indicator changes its color in response to the chemical reduction of the medium resulting from living cells. The results are expressed as relative fluorescence units (RFU).

The supernatant of each well was collected, and centrifuged to remove particulates, to be used to quantify Lactate Dehydrogenase (LDH, detection kit, Roche diagnostics, IN, USA). Cell lysate was also collected to measure Fibroblast Growth Factor (FGF, Omnikine ELISA kit, Assay Biotechnology, CA, USA) and Collagen type 1 (COL1, ELISA kit, FineTest, Wuhan, China) production. The LDH test was performed following manufacturer’s instruction and LDH concentration was spectrophotometrically read at 490/655 nm and it was reported in function of cell viability. The FGF and the COL1 immunoenzymatic tests were performed following manufacturer’s instruction and absorbance was spectrophotometrically measured at 450 nm. The measured absorbance values were converted into FGF (pg/ml) and COL1 (ng/ml) by means of a calibration curve obtained from standard solutions.

In Figure 11(A), cell viability by Alamar blue test at 24 and 72 hours of culture was shown. Fibroblasts cultured in direct contact with the film proliferate regularly, as well as CTR-, without differences between groups, at both experimental times. LDH is an enzyme released in medium when cell membrane is damaged and it is a useful parameter studying cytotoxicity. The analysis of LDH results (Figure 11(B)) showed a low release of LDH, both at 24 and at 72 hs. LDH values in CTR+ were always significantly higher than in all other groups. COL I is the main component of Extra-cellular matrix (ECM), and its expression is a common marker of cells differentiation.

Fibroblasts play an important role in wound healing, synthesizing collagen, one of the main components of the extracellular matrix. FGF promotes cell proliferation, angiogenesis and endothelial cell migration, contributing to improve wound healing. The film significantly stimulated FGF (Fig. 11(C)) and COL1 (Fig. 11(D)) synthesis in comparison with CTR-. Moreover, COL1 reached significant higher value when compared to CTR-, while it was not detected in CTR+ group.

Example 23: Water Vapour Permeability (WVP)

The WVTR (Water Vapour Transmission Rate, g/hm²) refers to the rate of transmission of water vapour through the area of a flat sample (film) induced by the difference in vapour pressure between two specific surfaces, under specific temperature and humidity conditions. This measurement is performed as described in the article“Preparation and characterization of active gelatine-based films incorporated with natural antioxidants”, Jian-Hua Li et al., Food Hydrocolloids 37 (2014) 166-173. Appropriately sized glass vials containing 3g anhydrous calcium chloride (relative humidity (RH) = 0) are used. Silicone is placed on the mouth of the vial onto which the film being measured is adhered. The vials, after having been weighed, are placed inside a desiccator saturated with 50% relative humidity by saturated NaCl solution. The desiccator is placed at 30 °C: the vials are weighed every day for 12 days. By plotting the weight measurements made as a function of time, a linear trend is obtained: the interpolation of the graph provides the parameters of the line. The value of WVTR was calculated from the slope of the straight line divided by the surface of the tested film (for exemplary films of the invention, this value corresponds to 0.00013m²):

WVTR= G/tA = (G/t)A

Wherein:

G/t = slope of the line (expressed in g/h),

A = sample area in m² (equal to 0.00013m²).

The water vapour permeability (WVP) is the water vapour transmission rate through a flat film area induced by a vapor pressure between two surfaces under specific conditions of moisture and temperature or the ease of moisture for penetrating and passing through the hydrophilic portion of film (E. Hernandez, Edible coatings for lipids and resins, in: J.M. Krochta, E.A. Baldwin, M.O. Nisperos-Carriedo (Eds.), Edible Coatings and Films to Improve Food Quality, Technomic Pub Co, Lancaster Pa 1994, pp. 279-304.). The WVP value is calculated with the following formula:

WVP = WVTR/Dr

Wherein:

Dr corresponds to the vapour tension of a saturated solution of [Mg(N0₃)₂] at 25 °C equal to 1670 Pa (table value).

Water Vapor Permeability was evaluated for all the cellulose-based films.

Table 14: Water Vapor Permeability values of cellulose-based films

Table 14 reports the WVP values obtained from Equation reported above. The presence of snail slime into films composition strongly modifies the Water Vapor Permeability. Values decrease by one or two orders of magnitude as a function of S content. For example, the WVP values vary from 1.3-10 ¹⁰ to 6.5· 10 ¹² g m/s m² Pa when measured on E5 and E5S_100, respectively.

Water Vapor Permeability was also evaluated for some chitosan-based films. A peculiar trend as a function of composition (Fig. 12) was observed since CS 7030 A and CS 7030 L exhibit WVP values significantly smaller than the other films of the series. Interestingly, C SOL films exhibit lower permeability than C L and C_A films (*p < 0.05). Moreover, the WVP values of C_SOL films are not significantly affected by water addition. Example 24: Antimicrobial properties of the films

The antibacterial activity of the cellulose-based films was assessed in vitro by means of a standardized Kirby-Bauer (KB) diffusion test on Mueller-Hinton agar plate [EUCAST: The European Committee on Antimicrobial Susceptibility Testing, Breakpoint Tables for Interpretation of MIC s and Zone Diameters, Version 6.0, 2016. http://www.eucast.org] against both Gram positive and Gram negative bacteria. In particular, the following panel of Gram positive and Gram negative reference bacterial strains was selected: Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 9591). Bacterial suspensions were prepared in 5 mL of sterile 0,9% saline solution by picking few colonies from a blood agar plate, thereafter were adjusted to match the turbidity standard of 0.5 McFarland unit (corresponding to 1,5 x 10⁸ CFU/mL, Colonies Forming Unit). These working solutions were swabbed on the Mueller-Hinton agar plate and disk-shaped cellulose films (0= 6 mm, cut with a cork-borer) were laid down on Agar plate and the inhibition of bacterial growth was determined by measuring the diameter of the bacterial- free zone around the sample after 24 hours of incubation at 37 °C. Zone is measured from edge to edge of the clear area, by a transparent ruler.

According to reference procedures, disks containing gentamicin (GMN 10 pg) were used as controls [CLSI, 2016Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing; Twenty-fifth Informational Supplement, CLSIdocument M100-S25 (2015)]. All experiments were performed in duplicate and in different days.

Tables 15 and 16 report the mean diameter of the bacterial -free zone measured for each composition on Gram-Positive and Gram-Negative bacterial strains, respectively. All the films made only of cellulose did not show any antibacterial activity, while a halo around the disk-shaped samples appeared when the composition is enriched by a snail slime addition, clearly indicating its antibacterial effect.

Table 15: mean diameters of the inhibition zones for Gram-positive bacteria cultured on

Cellulose-based films

Table 16: mean diameters of the inhibition zones for Gram -negative bacteria cultured on

Cellulose-based films

Of note, the cellulose-based disks containing S at 30% displayed inhibitory zone for Pseudomonas aeruginosa, an opportunistic pathogen recognized as an important cause of cutaneous, corneal, and respiratory tract infections. The in vitro antibacterial activity of the chitosan-based films was evaluated against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC25922) selected as controls and representative strains for Gram-positive and Gram-negative bacteria. The effectiveness of samples to inhibit bacterial growth was assessed by a standardized Kirby- Bauer (KB) diffusion test on Mueller-Hinton agar plate and by measuring the bacterial -free zone around the disk-shaped samples (0= 6 mm) after 24 h of incubation at 37 °C [L. Forte et al., J, Inorg. Biochem, 178 (2018) 43-53] All experiments were performed on duplicate in different days.

Results are reported in Table 17. There was no difference between the antibacterial effects on the different microbial species.

Table 17: Ranges of the inhibition zone diameters (mm) measured for chitosan-based films. NA = not appearing

Although it is generally recognized that chitosan solutions have strong antibacterial activities [54], chitosan films did not inhibit bacterial growth in agar diffusion tests because chitosan in a film form is unable to diffuse through the surrounding agar media [55] The present results confirm this feature since both C_A and C L samples did not inhibit bacterial growth. As a consequence, the bacterial-free zones observed for S- containing films could be definitely ascribed to snail slime addition. Chitosan films prepared in acetic acid and directly in S showed antibacterial activity at the highest S content (CS 3070 A, C SOL and CS 7030 SOL) confirming the inhibitory role of snail slime. On the contrary, films prepared in lactic acid did not display antibacterial properties irrespective to S content.

Example 25: Sunscreen properties Barrier properties of the films against UV-Vis light were investigated by means of Cary 60 Uv-Vis spectrophotometer: spectra were acquired in transmittance mode in the UV-Visible range (from 200 to 800 nm) on films cut into 1 cm wide rectangular strips directly inserted into the sample holder. Transparency was evaluated by the following equation:

Transparency value = ^lo^^r60°

where Uoo is the transmittance at 600 nm and X is the thickness of the film (pm) measured as described above. ( Comparative analysis of blend and bilayer films based on chitosan and gelatin enriched with LAE (lauroyl arginate ethyl) with antimicrobial activity for food packaging applications. Haghighi, H., De Leo, R., Bedin, E., Pfeifer, F., Siesler, H. W., & Pulvirenti, A. 19, 2019, Food Packaging and Shelf Life , pp. 31-39).

Spectra recorded on E5-based films are reported in Figure 13 as example.

Addition of snail slime produces films (E5S 7030, E5S 3070 and E5S 100) with barrier properties against UV light, even if it is added in the lower amount. In fact, as it can be observed in Figure 13, the spectra collected from films containing snail slime show a value of T% equal to 0 between 200 and 280 nm (UV region), suggesting they screen from UV radiation. Table 18 reports the transparency values: films with a transparency value under 5 can be considered transparent. Addition of snail slime maintains transparency while also conferring to the films UV screen properties.

Table 18: transparency values for the cellulose-based films

These properties (poor water vapor permeability of Example 23, antimicrobial activity of Example 24 and UV barrier of Example 25) allow a possible application of CMC -based films for food packaging as shown in Figure 14. In particular, films for food packaging ideally preserve/protect food from sunbeams, especially from UV radiation, which has sufficient energy to alter food.

Example 26: Development of drug-loaded films

100 mg of the antimicrobial drug Fluconazole (FL) were dispersed in 14 ml of snail slime containing glycerol (30% w/w on the gelatin weight) and NaOH (10M) to adjust the pH at 4,5, under magnetic stirring until complete drug solubilization. This solution was then mixed with the porcine gelatin solution solubilized in water (at 5% w/V) at 38°C.

After about 2 hours of stirring, 8.6 mL of this solution were poured in polyethylene Petri dishes (0= 5.5 cm) and put under laminar hood at room temperature overnight. The obtained films were stored at room temperature between two sheets of plastic-coated aluminum closed inside PVC bags.

The in vitro permeation studies were carried out in a Franz Cell system using 12 mL of receptor medium (phosphate buffer with 20% of ethanol w/V (pH 5.5)), maintained at a temperature of 32± 0.5 °C through thermostatic bath circulation and constantly stirred at 350 rpm during the experiments. In the donor compartment, circular films (0=1.1 cm) were placed on pig ear full thickness skin used as membrane. Each membrane was conditioned for 15 minutes in phosphate buffer solution and carefully placed in the interface between the donor and receptor compartments. Aliquots of 100 pL were collected from the receptor at 120, 240, 360, 480 and 1440 minutes (since the film is particularly intended for single daily application). Sink conditions were maintained with the replacement of the same volume of receptor medium. All collected samples were analyzed by HPLC-UV/Vis and Fluconazole quantification was obtained by the regression equation obtained from a standard curve prepared on the same day as the analysis. The cumulative amount of Fluconazole permeated through the pig ear dermis was calculated considering the film area (mg/cm²) and then plotted as a function of time (hours). Each sample was analyzed in quintuplicate and the data are expressed as mean ± SD. For comparison, 0.5 mL of drug solution (containing the same amount of FL loaded into the film), prepared dissolving the drug in phosphate buffer solution with 20% of ethanol w/V was added to the donor chamber. The results, reported in Figure 15, evidence that the film formulation enhances the drug permeation into the skin layer with respect to a drug solution containing ethanol, which is known for its penetration enhancement property. Thus, the film containing the biopolymer and the snail slime represents effective drug delivery systems to the skin.

The in vitro antimicrobial activity of drug-loaded gelatin films was evaluated against Candida albicans (ATCC 10231) and clinical isolates (F. Bonvicini et ah, Molecules , 2019, 24, 372): C. albicans C. glabrata, C. parapsilosis, C. tropicalis and C. krusei, selected since they represent the main systemic fungal infections. The effectiveness of samples to inhibit fungi growth was assessed by a standardized Kirby-Bauer (KB) diffusion test on Mueller- Hinton agar plate and by measuring the fungal-free zone around the disk-shaped samples (0 = 6 mm) after 24h of incubation at 37°C \L Forte, P. Torricelli, F. Bonvicini, E. Boanini, G.A. Gentilomi, G. Lusvardi, E. Della Bella, M. Fini, E. Vecchio Nepita, A. Bigi, Biomimetic fabrication of antibacterial calcium phosphates mediated by Polydopamine, ./. Inorg. Biochem., 178, (2018), pp. 43 53] Antifungal activity was measured following the procedure reported above in Example 24.

Gelatin film containing glycerol were used as control (NO HALO). All experiments were performed on duplicate in different days. Unloaded gelatin film without the snail slime were used as control. The results are reported in Table 19.

Table 19. Diameters of inhibition (mm) ± standard deviation

The results demonstrate that the control did not display any inhibition effect, while the drug-loaded films exhibit a clear inhibition halo. Therefore, the drug-loaded films exhibit favorable biopharmaceutical properties enabling the FL release, while maintaining its in vitro activity (high prolonged and effective drug release and high in vitro bioactivity against Candida).

Example 27: Structural characterization X-ray diffraction patterns were carried out by means of a Philips X'Celerator diffractometer equipped with a graphite monochromator in the diffracted beam (20 range from 4° to 50°, step size = 0,06680°, 40s/step). CuKa radiation (40 mA, 40 kV, 1.54 A) was used. The Fourier transform infrared spectra were recorded using a Thermo Scientific Nicolet iSlO FTIR spectrometer equipped with an ATR sampling device that uses a Germanium diamond as element for internal reflection. Spectra were acquired at room temperature in absorbance mode from 2300 to 800 cm^-1 with a resolution of 2 cm^-1. TGA was carried out using a Perkin-Elmer TGA-7. Heating was performed in a platinum crucible in air flow (20 mL/min) at a rate of 10 °C/min up to 800 °C. Samples weights were in the range of 5-10 mg.

The X-ray diffraction patterns collected from chitosan films are reported in Fig. 16: films obtained in acetic acid (C_A) show two prominent reflections at about 9.2° and 12 20, together with a sharp peak at 19720 attributed to type II hydrated polymorph of chitosan acetate [W. Chang, et ak, Food Hydrocoil. 90 (2019) 50-61.]. Snail slime is a complex mixture of active ingredients and it is not easy to discriminate the effectiveness and the interaction of each component with the chitosan functionalities. However, the comparison of the patterns collected from snail slime-containing samples puts into evidence that the material becomes less crystalline on increasing the S content. In fact, CS 7030 A films show only two broad halos, centered at about 8° and 20° of 2Q, while only a broad halo centered at 20° of 2Q can be detected when the samples contain a greater amount of solution (CS 7030 A). The X-ray pattern of the chitosan films obtained in lactic acid (C L), reported in Fig. 16, evidences a poorly crystalline structure, with two broad reflections at about 6° and at about 2072Q.

S addition provokes an overall decrease of the intensity of the diffraction patterns. It can be hypothesized that, on S addition, chitosan-snail slime interactions outweigh chitosan- chitosan interactions, leading to loss of structural order and, consequently, to the observed significant reduction in crystallinity. In agreement, the XRD patterns of all the samples of the C SOL series display just a very broad halo centered at about 20° of 2Q.

In agreement with the X-rays patterns, the infrared absorption spectrum of C_A films displays a number of bands which can be ascribed to the hydrated polymorph of chitosan [C. Qiao, et ak, Carbohydr. Polym. 206 (2019) 602-608] In particular, the absorption band at about 1640 cm^-1 can be assigned to the C O stretching (amide I), whereas those centered at about 1540 and 1390 cm^-1 can be attributed to NWH bending (amide II) and CWN stretching, respectively [I. Laceta, et al., Carbohydr. Polym. 93 (2013) 339-346] Addition of S provokes a general broadening of the spectra, which assume characteristic features of the spectrum of S powder (see Fig. 16), where an intense absorption peak, probably due to the high content of allantoin and glycolic acid in the solution, appears at around 1712 cm^-1. This absorption band also appears in the spectra of composite films and its intensity increases on increasing S content. The resolution of the absorption bands centered at about 1072 cm^-1, associated to CWO stretching, decreases on increasing S content, suggesting interactions between the hydroxyl groups of chitosan and polar groups of S through hydrogen bonds [I. Laceta, et al., Carbohydr. Polym. 93 (2013) 339-346] By comparing IR spectrum of S with that reported in literature [C. Trapella, et al., Sci. Rep. 8 (17665) (2018) 1-10] it is clear that snail slime obtained by MullerOne contains a minor amount of proteins with respect to that obtained by a different method of extraction. It is hypothesized that the use of ozone during snail stimulation should induce a partial protein degradation, thus lowering the proteic component of the final extract.

In agreement with XRD results, the infrared absorption spectrum of C L films is quite different and resembles those reported in literature for chitosan films prepared in lactic acid [M. Bajic, et al., Carbohydr. Polym. 219 (2019) 261-268] Addition of S to C L films has a similar effect to that observed on C_A films, and the spectra are similar to those recorded for the C SOL series.

The thermal stability of chitosan films was assessed by TGA analysis in air. Results obtained for the different films are reported in Fig. 17 together with the thermal behavior of lyophilized S. C_A films display three steps of thermo-oxidative degradation [M. Lavorgna, et al., Carbohydr. Polym. 82 (2010) 291-298] The first one, in the temperature range 35- 160 °C, is attributed to the loss of absorbed water. The second one, between 160 °C and 460 °C and centered around 310 °C, corresponds to the chemical degradation and deacetylation of chitosan [S.F.Wang, et al. Polym. Degrad. Stab. 90 (2005) 123-131], while the third step, in the temperature range 460-700 °C, can be associated with the oxidative degradation of the carbonaceous residue formed during the second step. The thermogravimetric plot of C L differs from that of C_A in the first region, which shows two distinct weight losses in the range 37-240 °C, in agreement with the different structures evidenced by XRD and FT- IR data. The derivative plot of TGA (DTG) of freeze-dried S (Fig. 17) displays a weight loss centered at 190 °C, which accounts for about 70% wt. of weight loss, and further degradation steps between 300 and 800 °C, probably due to the degradation of residues. A very similar thermogravimetric plot is shown by C SOL, with just some shift of the degradation steps to higher temperatures. Water addition (CS 7030 SOL and CS 3070 SOL) causes just a reduction of the relative amount of the first weight loss. When S is added to the composition of C_A and C L, all the films display similar thermogravimetric plots to that of C SOL series: in particular, the thermal degradation starts at a temperature lower than that of pure chitosan films and the first mass loss, determined between 37 °C and 300 °C, accounts for about 35% wt. and 48% wt. for the 7030 and 3070 compositions, respectively. Moreover, no water loss was observed between 35 °C and 160 °C.

Claims

1. A film comprising at least one polymer and a material secreted by a gastropod, wherein said material is slime, mucus or gastropod extract, and wherein said material is fresh or lyophilised.

2. The film according to claim 1, wherein the film is solid and/or lacks a backing sheet and/or exhibits both elastic and plastic properties.

3. The film according to claim 1 or 2, wherein said polymer is natural, semi synthetic, synthetic and/or biodegradable.

4. The film according to any one of the previous claims, wherein said polymer is selected from the group consisting of: gelatine, chitosan, cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, an alginate salt, carboxyvinyl polymer, rubber, carrageenan, a hyaluronate salt, starch, keratin, acrylic polymer and a combination thereof.

5. The film according to any one of the previous claims, wherein said polymer is cross-linked, preferably said polymer is cross-linked with any one of: the material secreted by a gastropod, a hyaluronate salt, a further polymer with opposite charge to said polymer, citric acid, gallic acid, tannic acid or ferulic acid.

6. The film according to any one of the previous claims, wherein said gastropod is a snail or a slug.

7. The film according to any one of the previous claims, wherein said material secreted by a gastropod is snail slime.

8. The film according to any one of the previous claims, further comprising:

- one or more excipients, and/or

- a cosmetic and/or therapeutic agent, and/or

- a micro/nanoparticulate system.

9. The film according to claim 8, wherein said cosmetic and/or therapeutic agent is selected from the group consisting of: antifungal agent, antibacterial agent, antiprotozoal agent, antiviral agent, keratolytic agent, exfoliating agent, anti-inflammatory agent, analgesic agent, anaesthetic agent, antiseptic agent, antihistamine agent, anti-scabies agent, antioxidant agent, a proteolytic enzyme, natural alkaloid, agent for the treatment of onychophagy and agent that stimulates the proliferation of fibroblasts, and/or said micro/nanoparticulate system comprises mica, clay or montmorillonite particles, preferably said antifungal agent is fluconazole.

10. The film according to any one of the previous claims, characterised by any one or more of the following parameters: - stress at break (o ) from 0.1 to 100 MPa,

- elongation at break (e_b) from 1 to 2000%,

- Young modulus (E) from 0.1 to 6000 MPa,

- maximum stress (o_m) from 0.1 to 100 MPa,

- detachment force (F) equal to or higher than 1 N,

- transparency equal to or lower than 5, wherein transparency is measured as:

wherein Tboo is the transmittance at 600 nm and X is the thickness of the film,

- a value of T% measured between 200 and 280 nm equal to zero, wherein T% = f/Io x 100, where h = light transmitted from the sample and Io = light transmitted from the source, and/or

- an X-ray diffraction pattern comprising a broad halo at 20 from 15° to 25° measured using CuKa radiation.

11. A procedure for the preparation of a film comprising the steps of:

wherein said material is slime, mucus or gastropod extract.

12. The procedure according to claim 11, wherein said solvent is water, preferably when said polymer is soluble in acid environment, said water is acidified, preferably said water is acidified with hydrochloric acid, acetic acid, lactic acid or citric acid.

13. The film as defined in any one of claims from 1 to 10 obtainable by the procedure according to claim 11 or 12.

14. The film as defined in any one of claims from 1 to 10 or 13 for use as a medicament.

15. The film according to claim 14 for use in a method of prevention and/or treatment of: a dermatological disorder, a cutaneous wound, an aphtha, an infection, dermatitis, atopic dermatitis, radiotherapy dermatitis, eczema, rash, acne vulgaris, psoriasis, rosacea, a burn, a sunburn, an ulcer, a diabetic ulcer, a scald, onychomycosis, onychophagia and/or a periodontal disease, preferably said infection is fungal, bacterial or viral, preferably said infection is a vaginal infection, a buccal infection, a skin infection, a nail infection, a mucosal infection, a lip infection, a wound infection, preferably said viral infection is a vaginal herpes, a lip herpes or cold sores.

16. Use of the film as defined in any one of claims from 1 to 10 or 13 as a cosmetic product or for packaging food or for storing food.

17. Non-therapeutic cosmetic method for preventing and/or decreasing a skin imperfection, for hydrating the skin and/or for soothing skin or mucosa, said method comprising the administration and/or application of the film as defined in any one of claims from 1 to 10 or 13, preferably said skin imperfection is cellulite, a stretch mark, a wrinkle, a scar, a stain and/or redness of the skin.

18. A kit, or a foil, or a patch, or a mask, or a gauze, or a drug delivery system comprising the film as defined in any one of claims from 1 to 10 or 13, preferably said foil is a foil for storing and/or packaging food.

19. A system comprising at least a first film and a second film, said first film and said second film being as defined in any one of claims from 1 to 10 or 13, wherein preferably the polymer in the first film is different from the polymer in the second film.

20. A solution comprising a polymer and a material secreted by a gastropod, wherein said polymer is in a concentration equal to or greater than 0.1% w/V compared to the volume of said solution, wherein said solution comprises from 5% to 100% in terms of volume of said material secreted by a gastropod, wherein said material is slime, mucus or gastropod extract and wherein said material is fresh or lyophilised.

21. A kit, or a foil, or a patch, or a mask, or a gauze, or a drug delivery system comprising the solution as defined in claim 20, preferably said foil is a foil for storing and/or packaging food.