Therapeutic contact lenses loaded with mucin-based nanoparticles

Abstract

The invention relates to a contact lens reversibly functionalised with covalently cross-linked glycosylated mucin nanoparticles optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic and the use thereof in treating dry eye disease and in the controlled release of active ingredients, markers, biomolecules and / or pre / post-biotics The invention further relates to some methods for preparing contact lenses loaded with covalently cross-linked glycosylated mucin-based nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

Description

[0001] TITLE

[0002] THERAPEUTIC CONTACT LENSES LOADED WITH MUCIN-BASED NANOPARTICLES

[0003] DESCRIPTION

[0004] TECHNICAL FIELD

[0005] The present invention relates to contact lenses reversibly functionalised with covalently crosslinked glycosylated mucin nanoparticles optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic and the use thereof in treating dry eye disease and in the controlled release of active ingredients, markers, biomolecules and / or pre / post-biotics.

[0006] The subject matter of the invention further relates to some methods for preparing contact lenses loaded with covalently cross-linked glycosylated mucin-based nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

[0007] STATE OF THE ART

[0008] The treatment of eye diseases by systemic administration of drugs is very complicated, as the blood-ocular barriers often prevent drugs from reaching the visual system.

[0009] Topical administration, by contrast, is convenient since it is not invasive; however, the bioavailability of topical drugs is lower than 5% and this limits their use. The main obstacle to the topical administration of drugs to the eye is again the presence of various barriers, such as the epithelium, and ocular tissue dynamics, which control the concentration and entry of solutes into the eye. These barriers preclude an effective passage of many drugs and lead to minimal absorption of the administered dose. In order to remedy the reduced penetration and effectiveness of molecules applied topically, most of the formulations used contain a larger dose of the active ingredient. However, this aspect often leads to problems tied to toxicity, making necessary the development of more effective methods of administering drugs through the ocular route. A good solution for treating chronic eye diseases is the use of contact lenses; for this reason, they have recently been used as medical devices. Numerous studies have highlighted that contact lenses loaded with drugs / molecules favour an increase in bioavailability, mainly by increasing the time of retention of the drugs in the eye. Nanotechnology has had broad application in the field of ocular drug administration. Various types of nanoparticles, such as micelles, microemulsions, liposomes and polymeric nanoparticles, have been integrated with contact lenses through four main methods. The first involves loading nanoparticles into the contact lenses by means of the immersion method (soaking), whilst the second provides for mixing nanoparticles and materials for contact lenses before forming the lens. The third method consists in implanting rings loaded with nanoparticles into the contact lenses, whilst the fourth method provides for coating the surface of the lenses with nanoparticles. United States patent US7438411 B2 discloses contact lenses comprising tunable metal nanoparticles, useful for extinguishing near-infrared energy.

[0010] Korean patent KR102357782B1 regards a contact lens containing hyaluronic acid-cholesterol conjugate-based micellar nanoparticles filled with a drug.

[0011] United States patent application US20040241207A1 discloses nanoparticles of different materials, both synthetic and of natural origin, such as alginate or gelatine, which are encapsulated in a contact lens.

[0012] United States patent application US20100330146A1 relates to particles and liquids that are used to create layers of materials on a contact lens, so as to reduce the diffusion of a drug.

[0013] Canadian patent CA2487940C discloses a drug administration system comprising a contact lens in which nanoparticles having a particle size smaller than about 50 nm are dispersed, an ophthalmic drug nano-encapsulated in a material from which said ophthalmic drug is capable of diffusing and migrating through said contact lens and into the post-lens tear film when said contact lens is positioned on the eye.

[0014] Canadian patent CA2683467C relates to contact lenses with an integrated biosensor for continuous non-invasive monitoring of physiological glucose, the lenses including nanoparticles of ceramic or metallic materials or other polymers and used as a colorimetric sensor.

[0015] European patent EP3525885B1 relates to artificial tear compositions, artificial tear gel compositions, contact lens care compositions, contact lens wear treatment compositions, ophthalmological drug vehicle compositions and drug vehicle compositions for topical use comprising one or more non-ionic surfactants and two or more non-Newtonian viscosity enhancing excipients and one or more of a polyol and / or an electrolyte and methods of use thereof.

[0016] Some examples of the use of mucin in non-particle form, used to functionalise contact lenses of varying nature, are reported in the literature: Carolin A. Rickert et al. describe mucin in nonparticle form covalently bound to hydrophobic contact lenses. The mucin is not released by the lenses and there are no references to any active ingredients loaded onto the lens (ACS Appl. Mater. Interfaces (2020), vol. 12, p. 28024-28033 and Biomaterial Advances (2023) vol. 145, p. 213233).

[0017] Benjamin Winkeljann et al. (Adv. Mater. Interfaces (2017), vol. 4, 1700186) describe coatings of mucin in non-particle form on contact lenses and, in this case as well, no release of mucin is observed and there are no references to any active ingredients loaded onto the lens.

[0018] A. Baszkin et al. (Biomaterials, (1984), vol. 3, p. 175-179) describe the adsorption of mucin onto contact lenses functionalised with polyvinylpyrrolidone, but no pharmaceutical applications are present.

[0019] Finally, Zhang Jin et al., Journal of Material Chemistry (2013) vol. 1 , no. 35, p. 4388, describe a method whereby nanoparticles are covalently inserted into the lens matrix through the formation of amide bonds, leading to the complete immobilisation of the nanoparticles within the hydrogel network, as confirmed by the release experiments described therein.

[0020] None of the abovementioned documents describes a contact lens comprising covalently crosslinked glycosylated mucin nanoparticles, optionally loaded with at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, as is provided, in contrast, by the present invention. One of the main advantages deriving from the possibility of having a contact lens comprising glycosylated mucin nanoparticles compared to mucin in nonparticle form is that of combining the humectant and lubricating properties of the protein with the possibility of being loaded and releasing the compound loaded into the nanoparticles in a prolonged and localised manner, thus reducing the frequency of applications and consequently improving patient compliance. Moreover, the fact that the binding of the covalently cross-linked glycosylated mucin nanoparticles with the lens is of a reversible type allows the lens to act as a matrix for the controlled release of the nanoparticles themselves, which can subsequently interact with the ocular surface. Finally, the nanoparticle formulation can increase mucin stability with respect to the proteolytic enzymes present at the ocular level, such as hydrolase, transferase and translocase. This increase in stability is due to the fact that the mucin protein conformation changes when it is in nanoparticle form. In its free form, mucin has sites that are more greatly exposed, making them easily accessible to enzymes which can bind to the sites and induce degradation. When the mucin is instead organised in nanoparticles, the same sites are better shielded. Added to this advantage there is also an increase in the solubility of the loaded compound, thus preserving its therapeutic properties and functions. The possibility of loading molecules with different activities (e.g. anti-inflammatory and anti-microbial) in a single production process allows several functions to be performed simultaneously.

[0021] DEFINITIONS

[0022] Unless otherwise defined, all the terms of the art, notations and other scientific terms used here are intended to have the meanings commonly understood by those who are skilled in the art to which this description belongs. In some cases, terms with commonly understood meanings are defined here for the sake of clarity and / or for ready reference; the inclusion of such definitions in the present description should thus not be interpreted as representing a substantial difference compared to what is generally understood in the art.

[0023] The terms “comprising”, “having”, “including” and “containing” are to be understood as open terms (i.e. with the meaning “comprising, but not limited to”) and are to be considered as a support also for terms such as “to consist essentially of, “consisting essentially of”, “to consist of” or “consisting of”.

[0024] For all the ranges specified in the text, figures and claims of the present patent application, it is understood that the endpoints of these ranges are included, just as it is understood that all the values within said ranges are described. The terms “obtainable”, “obtained”, “obtainable directly from”, “obtained directly from” are considered equivalent.

[0025] The term "physiologically acceptable excipient" refers to a substance that is devoid of any pharmacological effect of its own and does not produce adverse reactions when administered to a mammal, preferably to a human being. Physiologically acceptable excipients are well known in the art and are described, for example, in the Handbook of Pharmaceutical Excipients, sixth edition (2009), incorporated herein by reference.

[0026] The acronym “PGM” stands for “pig gastric mucin”.

[0027] The acronym “BSM” stands for bovine submaxillary mucin.

[0028] The acronym “NPs” stands for covalently cross-linked glycosylated mucin nanoparticles.

[0029] The term “mucin” preferably means a class of highly glycosylated proteins, natural or recombinant, characterised by the presence of one or more domains rich in serine and threonine residues which bind O-glycans containing saccharides selected from N-acetylgalactosamine, N- acetylglucosamine, fucose, galactose, sialic acids, and mannose, with a total serine and threonine content greater than 10%, preferably between 12% and 45%, more preferably between 15% and 36%, relative to the total quantity of amino acids of the protein. By way of non-exhaustive example, this definition includes: (i) mucins of animal origin, such as, for example, pig gastric mucin, preferably type III), bovine submaxillary mucin and snail mucin; (ii) mucins obtained by means of recombinant expression technologies; and / or (iii) mucins encoded by genes belonging to the MUC family, including MUC2, MUC5AC, MUC5B, MUC6, MUC7, or MUC19 and other functional analogues; proteins not encoded by MUC genes but having structural domains similar to those of mucins such as, for example, lubricin, TIM proteins (T-cell immunoglobulin and mucindomain), podocalyxin, and other glycosylated proteins with analogous structural and functional characteristics. According to a particularly preferred aspect, the mucin is pig gastric mucin, even more preferably type III, bovine submaxillary mucin or snail mucin.

[0030] According to a particularly preferred aspect, a protein is referred to as highly O-glycosylated when over 40% of the molecular mass consists of O-linked glycans and / or when the sequence has >1 potential O-glycosylation site every 5-10 residues (e.g. for MUC2 >80% mass consisting of O- linked glycans) (Moniaux et al., 2001 DOI: 10.2741 / a778; Ju et al., 2011 DOI: 10.1007 / s10719- 011-9332-3).

[0031] According to another preferred aspect, the expression “mucin-like domain” (MLD) means a protein region rich in serine and threonine residues (typically >30-50%) and intensely O-glycosylated, often with glycan chains containing sialic acid.

[0032] Preferably, “highly glycosylated protein” means a protein whose molecular mass is made up of over 40% carbohydrate content, or which has more than one expected O-glycosylation site every 5-10 amino acids inside the MLD region. The expression “protein with a high abundance of mucin-like domains” preferably means a protein containing a number of mucin-like domains, or mucin-like domains representing over 30% of the total length of the amino acid sequence.

[0033] According to a preferred aspect, the NPs are produced according to international patent application WO2021 / 260525 of the same applicant. The covalently cross-linked glycosylated mucin nanoparticles used in the present invention have an average particle diameter between 100 and 400 nanometres (nm), preferably between 150 and 300 nm and even more preferably about 250 nm. The average particle diameter was measured by means of a transmission electron microscope (TEM) and also confirmed with DLS (Dynamic Light Scattering) methods.

[0034] The acronym “NPs-PGM” stands for covalently cross-linked glycosylated pig gastric mucin nanoparticles.

[0035] The acronym “NPs-BSM” stands for covalently cross-linked glycosylated bovine submaxillary mucin nanoparticles.

[0036] “Biomolecules” means nucleic acids, peptides, lipids and growth factors.

[0037] The term “pre / post-biotic” means prebiotics and postbiotics, preferably selected from indoles, indole derivatives, short-chain fatty acids, long-chain fatty acids, polyphenols, phenolic derivatives, phenazine, lactones and / or quinolones.

[0038] The acronym “FITC” stands for the compound “fluorescein isothiocyanate”.

[0039] The terms “markers” and “dyes” are used interchangeably and are considered synonymous. A marker of particular interest is a fluorophore, preferably selected from fluorescein isothiocyanate, rhodamine B and derivatives, Alexa Fluor ®, DAPI, BODIPY and derivatives, rose bengal and / or a near-infrared fluorophore, preferably a cyanine and even more preferably cyanine 5.5. In particular, rhodamine B and derivatives, belonging to the rhodamine class, are used in microscopy and flow cytometry to trace molecules in cells, for example to visualise the interaction between proteins. Alexa Fluor®, which belongs to the class of synthetic fluorescent dyes, is used in imaging techniques such as immunohistochemistry to visualise specific cell structures, such as the surface markers of immune cells. DAPI, 4',6-diamidino-2-phenylindole dihydrochloride, belonging to the class of fluorescent indoles, is used in fluorescence microscopy to stain cell nuclei. BODIPY, belonging to the class of boron dipyrromethenes, is used to label lipids and proteins in biological studies thanks to its intense fluorescence and high photostability, as in the tracing of lipid metabolism.

[0040] The acronym “PBS” stands for phosphate buffered saline.

[0041] “Contact lens” means a medical device applied directly on the outer surface of the eye to correct a person’s visual defects. They are used as an alternative to eyeglasses, since they assure greater freedom of movement. Furthermore, contact lenses may be used for cosmetic purposes or for therapeutic purposes to treat eye diseases. “Hydrogel-based soft contact lens” means, for example, a lens based on poly(2-hydroxyethyl methacrylate), N-vinyl-2-pyrrolidone (NVP), acrylamide (AAm), polyvinyl acetate (PVAc), polyacrylonitrile (PAN), polyvinyl alcohol (PVA) or polyacrylamide (PAAm).

[0042] “Silicone hydrogel-based soft contact lens” means, for example, a lens based on balafilcon A, lotrafilcon A, lotrafilcon B, galyfilcon A, comfilcon A, asmofilcon A, enfilcon A, senofilcon A, omafilcon A, omafilcon B, filcon IV, narafilcon A and B, samfilcon A, fanfilcon A, defilcon A, stenfilcon A, somofilcon A, asmofilcon A, delofilcon A, efanfilcon A, or alphafilcon A.

[0043] “Rigid gas-permeable contact lens” means, for example, a standard RPG (rigid gas-permeable) lens (Boston EO, Menicon Z), an Ortho-K lens (Orthokeratology) (Paragon CRT, Euclid Emerald), a bifocal or multifocal RGP lens (SynergEyes Duette Multifocal, GP Bifocal Essilor), an RGP lens for keratoconus (Rose K, Scleral lenses), a toric RGP lens (Boston ES, Fluoroperm Toric), a hybrid lens (SynergEyes A, ClearKone), an RGP lens for irregular cornea (KeraSoft IC, Zenlens), an extended wear RGP lens (Menicon Z Night, Paragon HDS 100), a scleral lens (BostonSight SCLERAL, PROSE), or an RGP lens for myopia control (specific Ortho-K).

[0044] The expressions “contact lens coated with covalently cross-linked glycosylated mucin nanoparticles” and “contact lens comprising covalently cross-linked glycosylated mucin nanoparticles” are considered interchangeable and each of them means a contact lens in which the covalently cross-linked glycosylated mucin nanoparticles can reversibly coat the contact lens and / or can be reversibly deposited on the contact lens without forming a covalent bond. Said covalently cross-linked glycosylated mucin nanoparticles may be optionally loaded with at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post- biotic.

[0045] The expressions “contact lens reversibly coated with covalently cross-linked glycosylated mucin nanoparticles” and “contact lens comprising reversibly associated covalently cross-linked glycosylated mucin nanoparticles” are considered interchangeable and each of them means that the bond between the covalently cross-linked glycosylated mucin nanoparticles and the contact lens is reversible, i.e. is not of the covalent type.

[0046] The acronym “GA” stands for contact angle.

[0047] The acronym “EWC” stands for equilibrium water content.

[0048] “Room temperature” means a temperature between 20 and 25° C.

[0049] “Controlled release” means the constant, prolonged release of active ingredients loaded into or deposited on the surface of biomaterials, usually in specific areas, with the aim of maintaining a constant concentration of the active ingredient in the blood or in localised areas.

[0050] “Lens of the invention” or “contact lens of the invention” means a contact lens comprising covalently cross-linked glycosylated mucin nanoparticles, wherein said covalently cross-linked glycosylated mucin nanoparticles are optionally loaded with at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic. The acronym “DED” stands for “dry eye disease”.

[0051] The acronym “CLIDE” stands for “contact lens-induced dry eyes”, that is, dry eye disease induced by prolonged use of contact lenses.

[0052] “Biomolecule” means a molecule preferably selected from nucleic acids, peptides, lipids and / or growth factors.

[0053] The expression “covalently cross-linked glycosylated mucin nanoparticles (further) comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic” or the expression “covalently cross-linked glycosylated mucin nanoparticles (further) loaded with at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic” or the expression “covalently cross-linked glycosylated mucin nanoparticles (further) loaded” means that the nanoparticles encapsulate at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic. The acronym “EE” stands for “entrapment efficiency”, calculated with equation 1 given in the experimental part.

[0054] SUMMARY OF THE INVENTION

[0055] The present invention relates to a contact lens comprising covalently cross-linked glycosylated mucin nanoparticles, wherein the term “mucin” preferably refers to a class of highly glycosylated proteins, natural or recombinant, characterised by the presence of one or more domains rich in serine and threonine residues which bind O-glycans containing saccharides selected from N- acetylgalactosamine, N-acetylglucosamine, fucose, galactose, sialic acids, and mannose, with a total serine and threonine content greater than 10%, preferably between 12% and 45%, more preferably between 15% and 36%, relative to the total quantity of amino acids of the protein. By way of non-exhaustive example, this definition includes: (i) mucins of animal origin, irrespective of the species or source, such as, for example, pig gastric mucin, preferably type III), bovine submaxillary mucin and snail mucin; (ii) mucins obtained by means of recombinant expression technologies; and / or (iii) mucins encoded by genes belonging to the MUC family, including MUC2, MUC5AC, MUC5B, MUC6, MUC7, MUC19 and other functional analogues; proteins not encoded by MUC genes but having structural domains similar to those of mucins such as, for example, lubricin, TIM proteins (T-cell immunoglobulin and mucin-domain), podocalyxin, and other glycosylated proteins with analogous structural and functional characteristics.

[0056] According to a particularly preferred aspect, the mucin is pig gastric mucin, even more preferably type III, bovine submaxillary mucin or snail mucin.

[0057] According to a preferred aspect, the covalently cross-linked glycosylated mucin nanoparticles reversibly coat the contact lens and, in particular, they can coat the contact lens without covalent bonds, or they can be deposited on the contact lens without forming a covalent bond.

[0058] According to a further preferred aspect, the covalently cross-linked glycosylated mucin nanoparticles that coat the contact lens comprise at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

[0059] The subject matter of the present invention also relates to some methods for preparing the contact lens of the invention comprising covalently cross-linked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

[0060] The invention also relates to a contact lens comprising covalently cross-linked glycosylated mucin nanoparticles for use in treating dry eye.

[0061] Furthermore, the invention also relates to a contact lens comprising covalently cross-linked glycosylated mucin nanoparticles, reversibly associated with the lens itself, to enable the controlled release of the nanoparticles, which can be optionally loaded with at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic. Finally, the invention also relates to the use of covalently cross-linked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, for coating a contact lens, preferably selected from a hydrogel-based soft contact lens, a silicone hydrogel-based soft contact lens and a rigid gas- permeable contact lens.

[0062] BRIEF DESCRIPTION OF THE FIGURES

[0063] Figure 1 - figure 1 shows the transmittance spectra of contact lenses loaded with nanoparticles (NPs) at different concentrations. The samples are represented with the following symbols: circle (circles) for 0.20 mg / mL, square (squares) for 0.30 mg / mL, triangle (triangles) for 0.50 mg / mL, diamond (diamonds) for 0.75 mg / mL and hexagon (hexagons) for 1.00 mg / mL. The controls, corresponding to the lenses not loaded with NPs, are represented by asterisks. The figure shows that at nanoparticle concentrations lower than 1 mg / mL it is possible to obtain transmittance values of between 95% and 100%, within the visible spectrum, for the contact lenses loaded with nanoparticles.

[0064] Figure 2 - figure 2 shows the transmittance spectra of contact lenses with empty nanoparticles (NPs-PGM) or nanoparticles loaded with 200 pg / mL of dye (NPs-PGM + FITC) or loaded with 200 pg / mL of a beta blocker drug (NPs-PGM + atenolol). The figure shows that the transmittance of the empty contact lenses or the ones loaded with 200 pg / mL of a dye or drug, is in a range of between 97.5% and 100%.

[0065] Figure 3 - figure 3 shows the release velocity of a beta blocker drug (timolol) measured in the system of a lens without NPs (triangles) and lenses with NPs containing the drug (circles). The curve shows a reduction of two orders of magnitude in the release velocity in the system of contact lenses loaded with NPs containing the drug, compared to other release methods. Figures 4A and 4B - These figures show the cumulative release of covalently cross-linked glycosylated mucin nanoparticles (NPs) from contact lenses immersed in a suspension of NPs for 1 , 2 or 3 days, measured at 4 °C (A) and 25 °C (B).

[0066] Figure 5 - This figure shows the cumulative release from the contact lenses (loaded by immersion for 1 day) with reference to: (i) unloaded mucin-based NPs (ii) methylene blue encapsulated in NPs (API in NPs) and (iii) free methylene blue (free API).

[0067] DETAILED DESCRIPTION OF THE INVENTION

[0068] The subject matter of the present invention relates to a contact lens comprising covalently crosslinked glycosylated mucin nanoparticles (NPs), reversibly associated with the lens itself, optionally further comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

[0069] The mucin-based nanoparticles, as such or loaded with at least one further compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, are non-covalently (reversibly) applied to the contact lens at variable concentrations so as to enable the controlled release of the NPs over time, and this coating of mucin-based NPs endows the lens with lubricating properties as well as ones reinforcing the ocular mucosal barrier. Furthermore, if the NPs are loaded, one will observe a gradual release of drugs or bioactive molecules from the nanoparticles themselves when detached from the contact lens and even when still associated with the contact lens comprising them, thus enhancing the stability and effectiveness of the eye treatment. Therefore, among other things, the invention also resolves the problem of insufficient lubrication in contact lenses, including therapeutic ones, and the difficulty of maintaining the nanoparticles stable in the eye, if used as such as a method of topical ocular release of active ingredients.

[0070] According to a preferred aspect, the quantity of nanoparticles released by the contact lenses increases as the loading time increases, where loading preferably takes place at room temperature (preferably 25 °C). In particular, immersion times comprised between 5 hours and 7 days, preferably between 24 and 72 hours, in a suspension containing nanoparticles lead to a progressive increase in the material deposited and thus in the subsequently releasable amount. Such conditions enable efficient, reproducible loading, which may be modulated as a function of time.

[0071] According to another preferred aspect, once the loading time, preferably 24 hours, has been fixed, the release of nanoparticles will largely depend on the temperature, which may vary between 4 and 37 °C. Higher temperatures (> 25 °C) favour a more rapid, substantial release, often characterised by an initial “burst” phase followed by a more gradual release. Lower temperatures (4 °C), by contrast, strongly limit the release by slowing down diffusion phenomena and contributing to a greater stability of the system over time. According to another preferred aspect, the mucin of the covalently cross-linked glycosylated nanoparticles is selected from highly glycosylated proteins, either natural or obtained by recombinant methods, characterised by the presence of one or more domains with a high density of serine and threonine residues modified with O-glycans containing saccharides selected from N-acetylgalactosamine, N-acetylglucosamine, fucose, sialic acids, and mannose, with a total serine and threonine content greater than 10%, preferably between 12% and 45%, more preferably between 15% and 36%, relative to the total quantity of amino acids of the protein. By way of non-exhaustive example, this definition includes: (i) mucins of animal origin, such as, for example, pig gastric mucin, preferably type III), bovine submaxillary mucin and snail mucin; (ii) mucins obtained by means of recombinant expression technologies; and / or (iii) mucins encoded by genes belonging to the MUC family, including MLIC2, MLIC5AC, MLIC5B, MLIC6, MLIC7, or MLIC19 and other functional analogues, as well as highly glycosylated mucin-like proteins not encoded by MUC genes, such as, for example, lubricin, TIM proteins (T-cell immunoglobulin and mucin-domain), podocalyxin, and other structural and functionally similar glycoproteins.

[0072] According to a particularly preferred aspect, the mucin is pig gastric mucin, even more preferably type III, bovine submaxillary mucin or snail mucin.

[0073] According to a further preferred aspect, the contact lenses comprising covalently cross-linked glycosylated mucin nanoparticles, optionally further comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, at a concentration of less than 1 mg / mL, show transmittance values of between 95% and 100% within the visible spectrum, preferably in a range of between 97.5% and 100%, even more preferably in a range of between 98% and 100%.

[0074] According to another preferred aspect, a contact lens according to the invention comprising covalently cross-linked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, shows a ratio between the mass of the nanoparticles and the mass of the lens between 1 :1000 and 1 :1 , preferably between 1 :100 and 1 :10, and even more preferably between 1 :90 and 1 :70. According to another preferred aspect, the contact lens comprising covalently cross-linked glycosylated mucin nanoparticles is selected from a hydrogel-based soft contact lens, a silicone hydrogel-based soft contact lens and a rigid gas-permeable contact lens.

[0075] In particular, a hydrogel-based soft contact lens that may be used for loading the covalently crosslinked glycosylated mucin nanoparticles optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic is for example a lens based on poly(2-hydroxyethyl methacrylate), N-vinyl-2-pyrrolidone (NVP), acrylamide (AAm), polyvinyl acetate (PVAc), polyacrylonitrile (PAN), polyvinyl alcohol (PVA) or polyacrylamide (PAAm). A silicone hydrogel-based soft contact lens that may be used for loading the covalently crosslinked glycosylated mucin nanoparticles optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, is for example a lens based on balafilcon A, lotrafilcon A, lotrafilcon B, galyfilcon A, comfilcon A, asmofilcon A, enfilcon A, senofilcon A, omafilcon A, omafilcon B, filcon IV, narafilcon A and B, samfilcon A, fanfilcon A, defilcon A, stenfilcon A, somofilcon A, asmofilcon A, delofilcon A, efanfilcon A, or alphafilcon A.

[0076] A rigid gas-permeable contact lens that may be used for loading the covalently cross-linked glycosylated mucin nanoparticles optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic is for example a standard RPG (rigid gas permeable) lens (Boston EO, Menicon Z), an Ortho-K lens (Orthokeratology) (Paragon CRT, Euclid Emerald), a bifocal or multifocal RGP lens (SynergEyes Duette Multifocal, GP Bifocal Essilor), an RGP lens for keratoconus (Rose K, Scleral lenses), a toric RGP lens (Boston ES, Fluoroperm Toric), a hybrid lens (SynergEyes A, ClearKone), an RGP lens for irregular cornea (KeraSoft IC, Zenlens), an extended wear RGP lens (Menicon Z Night, Paragon HDS 100), a scleral lens (BostonSight SCLERAL, PROSE), or an RGP lens for myopia control (specific Ortho- K).

[0077] According to a preferred aspect, the mass of the covalently cross-linked glycosylated mucin nanoparticles that can be comprised on the contact lens of the invention is comprised between 0.005 and 5 mg, preferably between 0.01 mg and 1 mg and even more preferably between 0.05 and 0.5 mg.

[0078] The covalently cross-linked glycosylated mucin nanoparticles (NPs), optionally further comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, coat the contact lens. In particular, the NPs, optionally loaded with at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, can be deposited on the contact lens without forming a stable bond (reversible deposition) so as to enable their release over time.

[0079] According to a preferred aspect, the concentration of the at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic loaded into the covalently cross-linked glycosylated mucin nanoparticles deposited on the selected contact lens is comprised between 1 pg / ml and 500 pg / ml, more preferably between 100 pg / ml and 350 pg / ml and even more preferably between 150 pg / ml and 250 pg / ml.

[0080] At the ocular level, mucins are present in the tear film and act as a barrier against pathogenic agents, thus reducing microbial colonisation. The presence of glycans enables mucins to “sequester” a considerable amount of water, which makes mucins excellent lubricants. This property is fundamental for maintaining the viscosity and surface tension of the tear film.

[0081] In dry eye disease (DED), alterations occur in the homeostasis of the tear film which result in altered mechanical interactions between the eyelids and eyeball during spontaneous blinking. In healthy individuals, the ocular surface is covered by a concentrated layer of proteins, mucins - membrane-tethered mucins, together with dispersed secreted mucins - which form the muco- aqueous layer of the eye’s protective tear film. Mucins are glycoproteins characterised by central repetitions, in tandem, of amino acids rich in serine, threonine and proline, where the serine and threonine residues serve as O-glycosylation sites. It is believed that these large, negatively charged glycoproteins are involved in the hydration, protection and lubrication of the ocular surface, while at the same time retaining antimicrobial proteins and resisting the adhesion of particles and pathogenic agents.

[0082] In patients with dry eye disease, a reduced biosynthesis or loss of functional mucins has been observed, and it is hypothesised that this might cause a defective lubrication underlying the physiopathology. Prolonged use of contact lenses can induce dry eye disease (contact lens- induced dry eyes - CLIDE).

[0083] Dry eye, unfortunately, is difficult to treat. Ointments and tear replacements are used to help in relieving dry eye symptoms, but the penetration of molecules at the ocular level is strongly influenced by the presence of various barriers and the defence mechanisms of the eye itself, such as blinking, tear production, and the outflow of substances through the nasolacrimal duct.

[0084] The contact lenses of the invention, modified with the covalently cross-linked glycosylated mucin nanoparticles as such, resolve the problem due to DED and CLIDE thanks to the release of the mucin nanoparticles present on the lenses and compensate for the mucin deficiency characteristic of patients with DED. The release of mucin nanoparticles restores the lubrication and barrier system characteristic of the normal eye. Such effects are closely dependent on the biochemical properties of mucin; therefore, the lubricating and mucosal barrier reinforcement effect can also be restored when the NPs are optionally loaded with at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, since such compounds do not modify the intrinsic properties of mucin. Furthermore, the presence of the contact lens enables a continuous release of the mucin-based material for the whole time in which the lens is worn, thus overcoming the problem of the reduced penetration and effectiveness of molecules applied topically to the eye.

[0085] Therefore, the subject matter of the invention also relates to a contact lens as defined above comprising covalently cross-linked glycosylated mucin nanoparticles for use in treating dry eye. According to another preferred aspect, the contact lens of the invention comprises covalently cross-linked glycosylated mucin nanoparticles (NPs) comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

[0086] Notwithstanding the effectiveness of the medical therapies used to treat the aforementioned eye diseases, the present system of administration, mainly through eye drops and collyriums, poses various limits and disadvantages. Administering ophthalmic drugs to target tissues in the eye is difficult due to delivery barriers. The rapid lacrimal elimination of a drug instilled as eye drops for the treatment of anterior segment diseases results in a low ocular permeability of 1-5%. The blood-retinal barrier and clearance mechanisms that eliminate drugs from tears and from eye tissue make the systemic or topical administration techniques for delivering drugs into the posterior part of the eye ineffective. Although the hydrophilic corneal stroma mainly allows the diffusion of hydrophilic molecules, the administration of many effective hydrophobic drugs is considerably hindered.

[0087] Advantageously, the contact lens of the invention comprising covalently cross-linked glycosylated mucin nanoparticles, capable of being loaded with a compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic with different physicochemical properties, allows these compounds to be released slowly into the eye, with a treatment coverage that is prolonged over time with a consequent decrease in the number of daily treatments compared to traditional medicated eye drops, which require multiple daily instillations.

[0088] In addition, the covalently cross-linked glycosylated mucin nanoparticles can also be stably loaded with hydrophobic drugs, obtaining stable suspensions that are deposited on the contact lens.

[0089] Furthermore, the mucin nanoparticles have demonstrated to be mucoadhesive, thus enabling, once they have been released from the contact lens, a prolonged retention time in mucosal areas and favouring greater localised effectiveness and reduced systemic effects.

[0090] Therefore, the contact lens of the invention comprising covalently cross-linked glycosylated mucin nanoparticles (NPs), optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, is useful for the controlled release of at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

[0091] The covalently cross-linked glycosylated mucin nanoparticles used for coating a contact lens according to the invention can encapsulate a large variety of active ingredients. Such nanoparticles, reversibly associated with the lens surface, can be gradually released onto the eye, where they free the at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic in a controlled manner over time.

[0092] In particular, the following can be used: a) active ingredients used in treating dry eye disease, normally administered as ophthalmic solutions or nasal sprays, such as, for example, loteprednol etabonate, prednisolone, tacrolimus, perfluorohexyloctane, varenicline and / or lifitegrast; b) active ingredients used in treating eye infections and inflammations such as, for example, keratitis. Keratitis is an inflammatory process affecting the cornea, which represents the main defence barrier of the eye, and may heal without sequelae (surface forms) or leaving areas of the cornea which are not perfectly transparent, defined as “corneal leukomas” (deep forms). In relation to their aetiology, the types of keratitis may be divided into “infectious keratitis” and “non- infectious keratitis”, which may be of the ulcerative and non-ulcerative type. Among the ulcers of an infectious type, we distinguish between bacterial (Pseudomonas and Staphylococcus Aureus), fungal (Candida), viral (Herpes Simplex Virus) and protozoan (Acanthamoeba) forms. In the presence of a non-infectious ulcer, on the other hand, an autoimmune, inflammatory or neurotrophic disorder must be suspected. If not properly treated, keratitis can be complicated by loss of corneal tissue with the consequent formation of an ulcer and risk of perforation. The severity can be variable, and the aetiological diagnosis is essential, especially in the forms affecting the central cornea, where a suitable and timely treatment has the aim of preventing and limiting a permanent visual deficit. The therapy varies according to aetiology and has the objective of controlling inflammation and infection and of favouring the regrowth of the corneal epithelium. The therapy for bacterial keratitis is represented by the use of broad-spectrum antibiotics administered topically and / or orally, as a monotherapy or in association; in some cases, topical cortisone is relied on if necessary to reduce the inflammation. In mycotic keratitis, the therapy provides for the use of topical antimycotics in association with systemic antimycotics; topical and systemic antivirals are used instead as therapy in viral forms of keratitis. Finally, in the forms caused by Acanthamoeba, the conventional antibiotic therapy is not effective and particular eye drops (PHMB and biguanides) are used. The therapy must be continued for a long time, even for 6 -12 months. Re-epithelisation is obtained by using lubricants, therapeutic contact lenses, or by eyelid closure with bandages or surgical closure. The active ingredients for the treatment of eye infections and inflammations used for topical application and which can be loaded into the covalently cross-linked glycosylated mucin nanoparticles used for coating the contact lens of the invention can be antibacterial compounds, such as antibacterials belonging to the class of quinolones or fluoroquinolones, e.g. tobramycin, ciprofloxacin 0.3%, and / or levofloxacin 0.3%; antiviral compounds, e.g. idoxuridine, acyclovir, vidarabine, ganciclovir and / or trifluridine; antiinflammatory compounds, e.g. steroidal anti-inflammatory compounds such as betamethasone, dexamethasone, prednisolone and / or loteprednol; and non-steroidal anti-inflammatory compounds (NSAIDs), e.g. diclofenac, ketorolac and / or flurbiprofen; antiallergic compounds, e.g. antihistamines such as antazoline and / or levocabastine; and such as, for example, mast cell stabilisers such as lodoxamide and / or olopatadine; antiprotozoal compounds, e.g. those belonging to the class of azoles such as voriconazole; and immunosuppressant compounds for the treatment of inflammatory processes; c) active ingredients used in treating glaucoma. Glaucoma is a chronic, progressive disease that affects the optic nerve and can lead to vision loss. The pathology is due to an increase in internal eye pressure and in a limited number of cases to a reduction in the blood supply to the optic nerve (the nerve responsible for transmitting visual information from the retina to the brain). The active ingredients for treating glaucoma used for topical application that can be loaded into the covalently cross-linked glycosylated mucin nanoparticles used for coating the contact lens of the invention can be p-adrenergic active ingredients, e.g. timolol and / or atenolol; o-adrenergic active ingredients, e.g. apraclonidine; active ingredients such as carbonic anhydrase inhibitors, e.g. acetazolamide; and active ingredients such as prostaglandin analogues, e.g. latanoprost; d) immunomodulatory active ingredients. The ocular surface is considered a compartment of the common mucosal immune system. It offers the possibility of modulating the physiological immune response on the ocular surface and effectively combatting inflammation, which compromises tear film stability and causes tear hyperosmolarity. The immunomodulatory active ingredients used for topical application that can be loaded into the covalently cross-linked glycosylated mucin nanoparticles used for coating the contact lens of the invention can be cyclosporine A, mycophenolate, tacrolimus, rapamycin and / or leflunomide.

[0093] The covalently cross-linked glycosylated mucin nanoparticles used for coating a contact lens according to the invention, in addition to active ingredients, can also encapsulate a marker, a biomolecule and / or a pre / post-biotic. Said nanoparticles, reversibly associated with the lens surface, can be gradually released onto the eye, where they free the active ingredient in a controlled manner over time.

[0094] A marker of particular interest is a fluorophore, preferably selected from fluorescein isothiocyanate, rhodamine B and derivatives, Alexa Fluor, DAPI, BODIPY and derivatives, rose bengal and / or a fluorophore in the near infrared, preferably a cyanine and even more preferably cyanine 5.5.

[0095] A biomolecule of particular interest is a molecule preferably selected from nucleic acids, peptides, proteins, lipids and / or growth factors.

[0096] A pre / post-biotic compound of particular interest is selected from indoles, indole derivatives, shortchain fatty acids, long-chain fatty acids, polyphenols, phenolic derivatives, phenazine, lactones and / or quinolones.

[0097] The subject matter of the present invention also relates to two methods for preparing the contact lens of the invention comprising covalently cross-linked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

[0098] Said methods of preparation, carried out at room temperature, make it possible to obtain the coating of the contact lenses with covalently cross-linked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

[0099] The first method of preparation is an immersion method which provides for the preparation of a contact lens of the invention containing covalently cross-linked mucin NPs reversibly introduced onto the surface of the contact lens. This method of preparation comprises the following step: a) total or partial immersion of the contact lens in an aqueous suspension of covalently crosslinked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, preferably wherein the ratio between the mass of the nanoparticles and the mass of the lens is comprised between 1 :1000 and 1 :1 , more preferably between 1 :100 and 1 :10, and even more preferably between 1 :90 and 1 :70.

[0100] According to a preferred aspect, when the contact lens is completely immersed in the aqueous suspension of step a), a step b) of shaking the contact lens is carried out.

[0101] According to a preferred aspect, the shaking of the contact lens immersed in the aqueous suspension of step b) can be continued for a time of between 5 hours and 7 days, preferably it is continued for a time of between 3 and 5 days.

[0102] According to another preferred aspect, when the immersion of the lens in the solution is partial, the aqueous suspension of step a) is deposited in the concave part, i.e. on the inner surface of the lens.

[0103] According to a further preferred aspect, the aqueous suspension of step a) has a volume of between 0.1 and 0.4 mL. According to another preferred aspect, when the contact lens is immersed partially in the aqueous suspension of step a), it is left in contact with the suspension for a time of between 5 h and 7 days, preferably between 12 h and 24 h.

[0104] Said method can further comprise a step of drying the contact lens after its removal from the suspension.

[0105] According to a preferred aspect, the aqueous suspension is prepared by suspending the covalently cross-linked glycosylated mucin nanoparticles, optionally loaded, in a saline solution, preferably comprising NaCI and / or NaHCOs, more preferably comprising 0.9% NaCI and 0.015% NaHCO3.

[0106] According to another preferred aspect, the aqueous suspension of covalently cross-linked glycosylated mucin nanoparticles, optionally loaded, in which the contact lens is totally or partially immersed, has a concentration between 0.1 mg / mL and 3 mg / mL, preferably between 1 mg / mL and 2 mg / mL, and even more preferably it is 1.5 mg / mL.

[0107] According to a preferred aspect, when the covalently cross-linked glycosylated mucin nanoparticles reversibly deposited on the selected contact lens are loaded with at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, the concentration of the at least one compound is comprised between 1 pg / ml and 500 pg / ml, more preferably between 100 pg / ml and 350 pg / ml and even more preferably between 150 pg / ml and 250 pg / ml.

[0108] According to a further preferred aspect, in step a) the contact lens is kept totally immersed for a period of between 5 hours and 7 days, preferably for 5 days.

[0109] Furthermore, according to another preferred aspect, before the drying step c), the lens can be rinsed with a 0.9% NaCI aqueous solution.

[0110] The subject matter of the invention also relates to a second method for preparing the contact lens of the invention, which is a spray deposit method, whereby the cross-linked covalent mucin nanoparticles are reversibly deposited on the polymeric surface of the contact lens. This method of preparation comprises the following step: a) depositing an aqueous suspension of covalently cross-linked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, onto a contact lens by nebulisation, preferably wherein the ratio between the mass of the nanoparticles and the mass of the lens is comprised between 1 :1000 and 1 :1 , more preferably between 1 :100 and 1 :10, and even more preferably between 1 :90 and 1 :70.

[0111] According to a preferred aspect, the contact lens is positioned in proximity to a nebuliser, preferably with a nozzle having a diameter of between 0.2 mm and 0.5 mm. According to another preferred aspect, the nebuliser is an airbrush sprayer. According to a further preferred aspect, the nebulised drops have a diameter of between 10 pm and 100 pm.

[0112] According to another preferred aspect, the contact lens is positioned in proximity to the nebuliser indicated above at a distance of between 10 cm and 50 cm, more preferably between 15 and 25 cm.

[0113] According to another preferred aspect, the total duration of the nebulisation is comprised between 1 minute and 10 minutes, preferably between 2 and 5 minutes, and even more preferably it is 3 minutes . According to a further preferred aspect, the applications are carried out for 10 seconds at 10-minute intervals.

[0114] According to a further preferred aspect, the aqueous suspension is prepared by suspending the covalently cross-linked glycosylated mucin nanoparticles, optionally loaded, in a saline solution, preferably comprising NaCI and / or NaHCCh, more preferably comprising 0.9% NaCI and 0.015% NaHCO3.

[0115] According to another preferred aspect, the aqueous suspension of covalently cross-linked glycosylated mucin nanoparticles, optionally loaded, in which the contact lens is immersed has a concentration of between 0.1 mg / mL and 3 mg / mL, preferably between 1 mg / mL and 2 mg / mL, and even more preferably it is 1.5 mg / mL.

[0116] According to a preferred aspect, when the covalently cross-linked glycosylated mucin nanoparticles deposited on the selected contact lens are loaded with at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, the concentration of the at least one compound is comprised between 1 pg / ml and 500 pg / ml, more preferably between 100 pg / ml and 350 pg / ml and even more preferably between 150 pg / ml and 250 pg / ml.

[0117] The methods for preparing the lens according to the invention are advantageously associated with the preparation of the covalently cross-linked glycosylated mucin nanoparticles optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, which is carried out as described in international patent application WO2021 / 260525 of the same applicant.

[0118] The contact lens comprising covalently cross-linked glycosylated mucin nanoparticles optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, has various advantages tied to the presence of the covalently crosslinked glycosylated mucin nanoparticles. In fact, thanks to their reversible inclusion on the lens, the nanoparticles can be gradually released onto the ocular surface, where they perform a combined action: on the one hand, they favour the lubrication and reinforcement of the ophthalmic mucosal barrier, by exploiting the intrinsic properties of mucin in particle form; on the other hand, they enable a prolonged, controlled release of at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic. In particular, by means of the covalently cross-linked glycosylated mucin nanoparticles it is possible to load active ingredients of both a hydrophobic nature and hydrophilic nature onto the lens, thereby increasing, in particular, the solubility of hydrophobic active ingredients.

[0119] Normally, one observes a reduced absorption of topical ophthalmic active ingredients due to mucosal barriers and lacrimation. In order to overcome this problem, nanoparticles, nanopillars or layers of artificial materials are used for the prolonged release of drugs. Some materials, not present naturally in the eye (such as chitosan) are exploited to improve the mucoadhesion of the nanoparticles and hence the release of the drug. Advantageously, using instead the lens of the present invention, which comprises covalently cross-linked glycosylated mucin nanoparticles released over time, ensures maximum mucoadhesion at the site of release, thereby minimising the flushing out of the active ingredient (or of the dyes, biomolecules or pre / post-biotics) from the ocular site and prolonging the release time thereof.

[0120] It should also be considered that the use of contact lenses, including therapeutic ones for the release of drugs, can generally generate a decrease in the production of ocular mucins, and thus reduce lubrication, possibly leading to the pathological condition of dry eye disease. Therefore, one normally tries to reduce the risks of the pathological development of dry eye disease by associating lubricating eye drops with the lenses. Advantageously, using the lens of the present invention, which comprises covalently cross-linked glycosylated mucin nanoparticles, allows for the release of nanoparticles from the lens, thus enabling ocular lubrication by exploiting the natural method of lubrication of the eye, without the need to associate any eye drops.

[0121] The silicone materials of contact lenses are not materials normally present in the eye and may have surface properties very different from those of the surrounding biological environment, which may alter the homeostasis of the ocular surface. In order to resolve this problem, use is normally made of materials with properties that are more similar to human tissues to coat silicone lenses, such as nanoparticles produced with materials of natural origin, such as, for example, polysaccharides, though they are not naturally present in the eye. Advantageously, the contact lens of the present invention comprises covalently cross-linked nanoparticles of glycosylated mucin, which is a material already naturally present in the eye.

[0122] It should be considered that the use of contact lenses can trigger infections and inflammations, which are normally treated with antibiotics and lubricants via eye drops or with therapeutic contact lenses specifically developed for the release of antibiotics or anti-inflammatories. Advantageously, the contact lens of the lens of the present invention can naturally reduce bacterial enzymatic activities and has immunomodulating properties, by virtue of the coating of covalently cross-linked glycosylated mucin nanoparticles. The contact lens of the present invention is thus in itself more resistant to infections and inflammatory processes compared to other materials, while maintaining the possibility of being loaded with both antibiotic and antiinflammatory drugs.

[0123] Generally, the use of eye drops can pose problems: it is associated with an increase in the risk of infections, there may be poor patient adherence to the treatment plan and there may be dosage difficulties. Therefore, in order to overcome the problem, antibacterial and / or bacteriostatic excipients are used in eye drops, rigorous rules are applied for the administration of eye drops (single-dose products, storage at a controlled temperature, limited product shelf life), or a larger dose of the drug is prescribed to compensate for the reduced control over the autonomous administration of the patient. Advantageously, the contact lens of the invention enables an administration through a release that is prolonged over time thanks to the mucoadhesive properties of mucin. This reduces the patient’s intervention to a minimum, thereby decreasing the risk of infections, improving adherence to the treatment plan and controlling the dose.

[0124] The drugs utilised for topical ocular use have different chemical properties and often include hydrophobic molecules, which are easy to expel from the site of application because of lacrimation. Normally, the methods for producing nanoparticles only allow for a limited loading of hydrophobic active ingredients. Therefore, the most common standard method for solving the problem is to increase the dose of the hydrophobic active ingredient to make up for the low permeability and high rate of flushing away from the site. Advantageously, the method of the present invention for producing the lens enables a lens coated with nanoparticles loaded with both hydrophilic and hydrophobic active ingredients to be easily made. The hydrophobic active ingredients encapsulated in the covalently cross-linked glycosylated mucin nanoparticles, present in the contact lens of the invention show to be stable and are released in a prolonged manner over time.

[0125] Normally, there are difficulties in production and scalability of the processes for producing lenses coated with nanoparticles. Therefore, complex nanoparticle production methods are used, which make it difficult to directly integrate them with the lens coating process. The method generally used to create the layer of nanoparticles on a lens is the immersion method, which takes a long time. Advantageously, the lens of the invention is produced in only two steps connected to each other: one-pot synthesis of the nanoparticles according to the process described in international patent application WO2021 / 260525 of the same applicant and the direct deposition thereof on the lens. The process of depositing the nanoparticles on the lens can be carried out with reversible deposition methods, specifically with the standard immersion method or with the spray deposition method (which takes a shorter time).

[0126] A personalised self-medication therapy can be carried out with over-the-counter drugs, remedies without a doctor’s prescription or other personal care practices: each patient needs different doses and one or more different drugs. Generally, therefore, in order to resolve this problem, the nanoparticles are bound to a specific lens and there are ranges of lenses on the shelf to cover different treatment plans, which, however, are not really personalised. Advantageously, the method of producing the contact lens of the invention enables personalised, tailor-made ocular formulations to be prepared as needed for home application.

[0127] Furthermore, it should be considered how high the cost of the proteins for the usual development of nano-systems is. Normally, in order to balance costs and biocompatibility, the use of nonprotein materials of natural origin is relied on. Advantageously, the contact lens of the invention contains nanoparticles of mucin, which is a very low-cost protein compared to others of animal origin.

[0128] A further advantage of the present invention is that the method for producing the contact lens of the invention is adaptable to the different standard coating methods and the process has a modest cost and does not require complex machinery.

[0129] EXAMPLES

[0130] Example 1 of contact lenses loaded with cross-linked mucin nanoparticles by means of the immersion method.

[0131] The following preparations from A) to B) were each made under the same conditions on several types of lenses: Comfilcon A, Balafilcon, Omafilcon A and B, Filcon IV.

[0132] A) Covalently cross-linked glycosylated pig gastric mucin nanoparticles (NPs-PGM)

[0133] A 1.5 mg / mL suspension of NPs-PGM was prepared in a 2 mL 0.9% NaCI aqueous solution. Then a contact lens was inserted into every vial and after that the vials were shaken with a rocking shaker, at room temperature and with a speed of 150 / 200 rpm for five days.

[0134] Then the lenses were washed a minimum of 3 times with a 0.9% NaCI solution to eliminate the excess nanoparticles.

[0135] After that, the lenses were removed from the solutions, which were subsequently analysed to determine the mass of NPs-PGM deposited on the lens. A calibration curve was constructed using a Hitachi UH5300 UV-Vis spectrophotometer in a range of concentrations of (0.001 ; 0.005; 0.01 ; 0.03; 0.05; 0.1 ; 0.2; 0.3 mg / mL). The mass of NPs-PGM deposited on the contact lens ranges between 0.01 and 1 mg.

[0136] B) Glycosylated covalently cross-linked bovine submaxillary mucin nanoparticles (NPs-BSM)

[0137] A 1 .5 mg / mL suspension of NPs-BSM was prepared in a 2 mL 0.9% NaCI aqueous solution. Then a contact lens was inserted into every vial and after that the vials were shaken with a rocking shaker, at room temperature and with a speed of 150 / 200 rpm for five days. Then the lenses were washed a minimum of 3 times with a 0.9% NaCI solution to eliminate the excess nanoparticles.

[0138] After that, the lenses were removed from the solutions, which were subsequently analysed to determine the mass of NPs-PGM deposited on the lens. A calibration curve was constructed using a Hitachi LIH5300 UV-Vis spectrophotometer in a range of concentrations of (0.001 ; 0.005; 0.01 ; 0.03; 0.05; 0.1 ; 0.2; 0.3 mg / mL).

[0139] The mass of NPs-BSM deposited on the contact lens ranges between 0.01 and 1 mg.

[0140] Example 2

[0141] Preparation of contact lenses loaded with covalently cross-linked glycosylated mucin nanoparticles (NPs) containing a dye (marker), an active ingredient and / or a pre / post biotic by means of the immersion method.

[0142] The following preparations from A) to C) were each made under the same conditions on several types of lenses: Comfilcon A, Balafilcon, Omafilcon A and B, Filcon IV.

[0143] A) Dyes (for example FITC or cyanine)

[0144] NPs-PGM or NPs-BSM loaded with a dye, e.g. fluorescein isothiocyanate (FITC), at a concentration of 200 pg / mL were suspended at a concentration of 1.5 mg / mL in a 2 mL 0.9% NaCI aqueous solution. Then a contact lens was inserted into every vial and after that the vials were shaken with a rocking shaker, at room temperature and with a speed of 150 / 200 rpm for five days.

[0145] Then the lenses were washed a minimum of 3 times with a 0.9% NaCI solution to eliminate the excess nanoparticles.

[0146] In order to determine the amount of FITC encapsulated inside the nanoparticles before they are deposited on the contact lens, Equation 1 was used, also using the calculation of the residual amount of dye in the supernatants. The analyses were conducted by fluorescence spectroscopy using a TCSPC Horiba Jobin Yvon Fluorolog 3 fluorimeter, equipped with a 450 W xenon lamp and a Hamamatsu R928 photomultiplier. In order to identify the excitation wavelength, the UV- Vis absorption and emission bands were used. Initially, the dye was excited at the excitation wavelength of the dye encapsulated in the NPs, thus 490 nm in the present case of the FITC dye, using the excitation slit 2, and a seven-point calibration curve was constructed. This curve was obtained by measuring the maximum signal of dye emission at 520 nm for the FITC dye, using the emission slit 4, for various dye concentrations, i.e.: 1 , 5, 10, 25, 50, 100 and 200 ng / mL.

[0147] The final concentration of FITC loaded into the covalently cross-linked glycosylated mucin nanoparticles deposited on the contact lens is about 200 pg / mL. Equation 1

[0148] (initial mass of the compound used - mass of the compound remaining in the supernatant)

[0149] EE initial mass of the compound used

[0150] The same procedure was also carried out with NPs loaded with cyanine, and a final concentration of cyanine loaded into the covalently cross-linked glycosylated mucin nanoparticles of about 200 pg / mL was obtained.

[0151] The mass of NPs-PGM or NPs-BSM loaded with a dye, either FITC or cyanine, and deposited on the contact lens ranges between 0.01 and 1 mg.

[0152] B) Beta-blocker active ingredients (for example atenolol)

[0153] NPs-PGM or NPs-BSM loaded with 200 pg / ml of a beta-blocker active ingredient, e.g. atenolol, were suspended at a concentration of 1.5 mg / mL in a 2 mL 0.9% NaCI aqueous solution. Then a contact lens was inserted into every vial and after that the vials were shaken with a rocking shaker, at room temperature and with a speed of 150 / 200 rpm for five days.

[0154] The active ingredient was quantified by HPLC-UV using an HPLC Varian Prostar system equipped with a 410 autosampler. As the mobile phase, (90% TAA - 10% ACN) was used, where the acetonitrile was used as the organic phase and the TAA, ammonium acetate buffer, as the aqueous phase; the mobile phase was delivered with a flow rate of 1 mL / min. A standard solution of the beta blocker drug was introduced into the HPLC-UV instrument, and the absorbance peak characteristic of the specific active ingredient was identified, i.e. at 224 nm in the case of atenolol. In order to quantify the drug, a calibration curve was constructed with eight points, namely 10, 25, 50, 75, 100, 150, 200 and 250 pM.

[0155] The final concentration of atenolol loaded into the covalently cross-linked glycosylated mucin nanoparticles deposited on the contact lens is about 200 pg / mL.

[0156] The mass of NPs-PGM or NPs-BSM loaded with a beta-blocker active ingredient, e.g. atenolol, deposited on the contact lens ranges between 0.01 and 1 mg.

[0157] C) Pre / post-biotic molecules (for example indoles, indole derivatives, short-chain fatty acids, long- chain fatty acids, polyphenols, phenolic derivatives, phenazine, lactones, quinolones)

[0158] NPs-PGM or NPs-BSM loaded with 200 pg / ml of a pre / post-biotic molecule, e.g. indole, were suspended at a concentration of 1.5 mg / mL in a 2 mL 0.9% NaCI aqueous solution. Then a contact lens was inserted into every vial and after that the vials were shaken with a rocking shaker, at room temperature and with a speed of 150 / 200 rpm for five days.

[0159] Then the lenses were washed a minimum of 3 times with a 0.9% NaCI solution to eliminate the excess nanoparticles.

[0160] The pre / post-biotic molecules were quantified by means of an LCMS-8045 liquid chromatographmass spectrometer (Shimadzu, Kyoto, Japan); it was used for the quantification method in the binary gradient mode. The gradients, columns and solvents are defined based on the specific molecule, starting from quantification data present in the literature. For the quantification of indole, a Luna C18 column (150 x 2.1 mm) with a positive polarity mobile phase composed of 0.005% TFA (H2O) to 0.005% TFA (ACN) was used. The mass spectrometer is equipped with an electrospray ionisation (ESI) source operating in a positive or negative ion mode, depending on the molecule considered. In the case of indole, the positive mode was used.

[0161] The final concentration of indole loaded into the covalently cross-linked glycosylated mucin nanoparticles deposited on the contact lens is about 200 pg / mL.

[0162] The mass of NPs-PGM or NPs-BSM loaded with a pre / post-biotic molecule, e.g. indole, deposited on the contact lens ranges between 0.01 and 1 mg.

[0163] Example 3 of contact lenses loaded with cross-linked mucin nanoparticles by means of the spray deposit method.

[0164] The following preparations from A) to C) were each made under the same conditions on several types of lenses: Comfilcon A, Balafilcon, Omafilcon A and B, Filcon IV.

[0165] A suspension of NPs-PGM was prepared at a concentration of 1.5 g / ml in a 2 mL 0.9% NaCI aqueous solution.

[0166] Then the suspension thus obtained was introduced into the reservoir of an airbrush sprayer (from 2 to 10 mL).

[0167] The contact lens was positioned vertically at a distance of 15-20 cm from the spray nozzle by means of tweezers. Optionally, the lens is rested on a rest surface, and the spray is positioned vertically to the lens.

[0168] The spray was activated at the minimum pressure (1 bar) and the flow of the nebulised suspension of NPs-PGM, oriented towards the contact lens, was maintained for a time ranging from 1 to 3 min.

[0169] The mass of NPs-PGM deposited on the contact lens ranges between 0.01 and 1 mg. mucin

[0170] A suspension of NPs-BSM was prepared at a concentration of 1.5 g / ml in a 2 mL 0.9% NaCI aqueous solution.

[0171] Then the suspension thus obtained was introduced into the reservoir of an airbrush sprayer (from 2 to 10 mL).

[0172] The contact lens was positioned vertically at a distance of 15-20 cm from the spray nozzle by means of tweezers. Optionally, the lens is rested on a rest surface, and the spray is positioned vertically to the lens.

[0173] The spray was activated at the minimum pressure (1 bar) and the flow of the nebulised suspension of NPs-BSM, oriented towards the contact lens, was maintained for a time ranging from 1 to 3 min. The mass of NPs-BSM deposited on the contact lens ranges between 0.01 and 1 mg.

[0174] Example 4

[0175] Preparation of contact lenses loaded with covalently cross-linked glycosylated mucin nanoparticles containing a dye (marker), an active ingredient and / or a pre / post biotic by means of the spray deposit method.

[0176] The following preparations from A) to C) were each made under the same conditions on several types of lenses: Comfilcon A, Balafilcon, Omafilcon A and B, Filcon IV.

[0177] A) Dyes (for example FITC or cyanine)

[0178] NPs-PGM or NPs-BSM loaded with 200 pg / mL of a dye, e.g. fluorescein isothiocyanate (FITC) or cyanine, were suspended at a concentration of 1.5 mg / mL in a 2 mL 0.9% NaCI agueous solution. Then the suspension thus obtained was introduced into the reservoir of an airbrush sprayer (from 2 to 10 mL).

[0179] The contact lens was positioned vertically at a distance of 15-20 cm from the spray nozzle by means of tweezers. Optionally, the lens is rested on a rest surface, and the spray is positioned vertically to the lens.

[0180] The spray was activated at the minimum pressure (1 bar) and the flow of the nebulised suspension of NPs loaded with a dye, fluorescein isothiocyanate (FITC) or cyanine, oriented towards the contact lens, was maintained for a time ranging from 1 to 3 min.

[0181] In order to determine the amount of dye (fluorescein or cyanine) encapsulated inside the nanoparticles, Eguation 1 , already given above in example 2, was used, also using the calculation of the residual amount of dye in the supernatants. The analyses were conducted by fluorescence spectroscopy using a TCSPC Horiba Jobin Yvon Fluorolog 3 fluorimeter, eguipped with a 450 W xenon lamp and a Hamamatsu R928 photomultiplier. In order to identify the excitation wavelength used the UV-Vis absorption and emission bands were used. Initially, the dye was excited at the excitation wavelength of the dye encapsulated in the NPs (490 nm in the case of the FITC dye, using the excitation slit 2), and a seven-point calibration curve was constructed. This curve was obtained by measuring the maximum signal of dye emission (at 520 nm in the case of FITC, using the emission slit 4) for various dye concentrations, i.e.: 1 , 5, 10, 25, 50, 100 and 200 ng / mL.

[0182] The final concentration of FITC (or cyanine) loaded into the covalently cross-linked glycosylated mucin nanoparticles deposited on the contact lens is about 200 pg / mL.

[0183] The mass of NPs-PGM or NPs-BSM loaded with a dye, i.e. FITC or cyanine, deposited on the contact lens ranges between 0.01 and 1 mg.

[0184] B) Beta-blocker drugs (for example atenolol)

[0185] NPs-PGM or NPs-BSM loaded with 200 pg / mL of a beta-blocker active ingredient, e.g. atenolol, were suspended at a concentration of 1.5 mg / mL in a 2 mL 0.9% NaCI agueous solution. Then the suspension thus obtained was introduced into the reservoir of an airbrush sprayer (from 2 to 10 mL). The contact lens was positioned vertically at a distance of 15-20 cm from the spray nozzle by means of tweezers. Optionally, the lens is rested on a rest surface, and the spray is positioned vertically to the lens.

[0186] The spray was activated at the minimum pressure (1 bar) and the flow of the nebulised suspension of NPs loaded with atenolol, oriented towards the contact lens, was maintained for a time ranging from 1 to 3 min.

[0187] The active ingredient was quantified by HPLC-UV using an HPLC Varian Prostar system equipped with a 410 autosampler. As the mobile phase, (90% TAA - 10% ACN) was used, where the acetonitrile was used as the organic phase and the TAA, ammonium acetate buffer, as the aqueous phase; the mobile phase was delivered with a flow rate of 1 mL / min. A standard solution of the beta blocker drug was introduced into the HPLC-UV instrument, and the absorbance peak characteristic of the specific drug was identified (for example, at 224 nm in the case of atenolol). In order to quantify the drug, a calibration curve was constructed with eight points ranging from 10 to 250 pM.

[0188] The final concentration of the beta-blocker active ingredient, e.g. atenolol, loaded into the covalently cross-linked glycosylated mucin nanoparticles deposited on the contact lens is about 200 pg / mL.

[0189] The mass of NPs-PGM or NPs-BSM loaded with an active ingredient beta-blocker, e.g. atenolol, deposited on the contact lens ranges between 0.01 and 1 mg.

[0190] C) Pre / post-biotic molecules (for example indoles, indole derivatives, short-chain fatty acids, lonq- chain fatty acids, polyphenols, phenolic derivatives, phenazine, lactones, quinolones)

[0191] NPs-PGM or NPs-BSM loaded with 200 pg / mL of a pre / post-biotic molecule, e.g. indole, were suspended at a concentration of 1.5 mg / mL in a 2 mL 0.9% NaCI aqueous solution. Then the suspension thus obtained was introduced into the reservoir of an airbrush sprayer (from 2 to 10 mL).

[0192] The contact lens was positioned vertically at a distance of 15-20 cm from the spray nozzle by means of tweezers. Optionally, the lens is rested on a rest surface, and the spray is positioned vertically to the lens

[0193] The spray was activated at the minimum pressure of 1 bar and the flow of the nebulised suspension of NPs loaded with indole, oriented towards the contact lens, was maintained for a time ranging from 1 to 3 min.

[0194] The pre / post-biotic molecules were quantified by means of an LCMS-8045 liquid chromatographmass spectrometer (Shimadzu, Kyoto, Japan); it was used for the quantification method in the binary gradient mode. The gradients, columns and solvents are defined based on the specific molecule, starting from quantification data present in the literature. For example, for the quantification of the indole, a Luna C18 column (150 x 2.1 mm) with a positive polarity mobile phase composed of 0.005% TFA (H2O) to 0.005% TFA (ACN) was used. The mass spectrometer is equipped with an electrospray ionisation (ESI) source operating in a positive or negative ion mode, depending on the molecule considered (in the case of indole, the positive mode was used). The final concentration of the pre / post-biotic molecule, e.g. indole, loaded into the covalently cross-linked glycosylated mucin nanoparticles deposited on the contact lens is about 200 pg / mL. The mass of NPs-PGM or NPs-BSM loaded with a pre / post-biotic molecule, e.g. indole, deposited on the contact lens ranges between 0.01 and 1 mg.

[0195] Example 5

[0196] Characterisation of lenses loaded with covalently cross-linked glycosylated mucin nanoparticles (NPs)

[0197] A) Contact angle

[0198] Measurements of the contact angle were made to measure the wettability characteristics of the contact lenses. The lenses analysed were obtained by totally immersing the lenses in an aqueous suspension of covalently cross-linked glycosylated mucin nanoparticles, wherein the ratio between the mass of the nanoparticles and the mass of the lens is 1 :83, for five days.

[0199] The lenses as such and lenses loaded with NPs were analysed using this technique with the aim of understanding whether, following a modification with the NPs, the surface of the contact lenses showed characteristics that were more hydrophilic or hydrophobic.

[0200] A Theta Lite optical tensiometer from Biolin Scientific, equipped with a Basler acA1300-200pm camera and tungsten carbide calibration ball, was used for the analysis. The tensiometer features precise droplet deposition, automated or manual, allowing the volume of the droplets to be maintained constantly equal in order to reduce any variation in the results to a minimum. The Theta Lite camera captures extremely rapid wetting phenomena, up to 2068 FPS, and performs exceptionally precise droplet shape analyses with a resolution of 1280 x 1024 pixel. The software used by the instrument is “OneAttension”. During the experiment, a calibration was first performed with the tungsten carbide ball; subsequently, the lenses were cut, since the surface of analysis had to be flat. The sessile drop (SD) technique, which entails positioning a droplet of liquid on a substrate of interest, was used.

[0201] Results of the measurements performed via the sessile drop method: contact angle of the contact lens (Comflicon A) as such (not treated with NPs) = 31 .7° contact angle of the contact lens loaded with Nps = 17.9°

[0202] The contact angle of the silicone hydrogel lens corresponds to the values stated among the technical features of the lenses and in previous studies (D0i:https: / / doi.org / 10.1016 / j.clae.2012.08.039).

[0203] The contact angle measured for the contact lenses with NPs has a lower value compared to the one without nanoparticles, indicating an increase in the hydrophilia of the lenses with NPs.

[0204] Therefore, a smaller contact angle (CA) means a better wetting capacity of the material on a substrate and thus a greater stability of the tear film distributed over the lens surface. Therefore, loading with NPs increases the wettability of the contact lens, which is a fundamental factor not only for the treatment of dry eye syndrome, but also for better eye movement and comfort.

[0205] B) Water content

[0206] Standard ISO 18369-4:2017, specific for contact lenses, defines water content as the mass fraction present in a hydrated material that is perfectly equilibrated at room temperature. It is an important parameter to define, because it is correlated to the degree of dehydration of the lens. Dehydration in turn influences lens parameters such as diameter, sagittal depth, base curve, and oxygen and ion permeability. Therefore, equilibrium water content (EWC) is one of the properties of the contact lenses that were investigated and was calculated using the formula in equation 2: Equation 2 100 where W1 indicates the weight of the wetted lens and W2 is the weight of the dry lens.

[0207] After the lenses had been weighed in the dry state, they were immersed in tear fluid and weighed at regular time intervals. The equilibrium water content was quantified as a percent variation (%) of the lens immersed in the solution relative to its dry weight. The equilibrium water content interval measured is between 18 and 29%, a result comparable with that of commercial lenses. The measurements were made by weighing the lens with a Sartorius analytical balance with a capacity of resolution up to 0.0001 g. As regards the dry contact lenses, measurements were made taking care to dry the lens with an absorbent cloth, whereas the wetted contact lens was rehydrated in distilled water at room temperature for 24 hours.

[0208] The weighing operations were carried out in triplicate for six different conditions, after five days’ soaking of all the lenses.

[0209] Table 1 below shows the values and relative standard deviations (RSD) for all the conditions for which the water content was calculated.

[0210] Table 1 From the table it may be deduced that the percentages of water content are similar for all the conditions described and there are no statistically significant differences compared to the unmodified contact lens. In particular, the lenses treated with nanoparticles (NPs) have a water content that ranges between 18% and 29%, in line with the values of commercial lenses. Therefore, as far as water content is concerned, the treated lenses fall within the criteria for allowing comfortable properties to be maintained.

[0211] Optical transparency, a fundamental factor in contact lenses, regards the ability to transmit the visible light spectrum; therefore, it is decisive for ensuring good vision. Studies suggest that materials such as hydrogels, used for contact lenses, should allow the passage of over 90% of the visible light spectrum. This property is also crucial in research involving the use of contact lenses to administer drugs.

[0212] Optical transparency experiments were carried out by UV spectrophotometry to verify that the lens-NPs system maintained the same characteristics of transparency as untreated lenses.

[0213] Empty NPs-PGM and NPs-PGM loaded with FITC or atenolol were loaded onto lenses and were then introduced into a cuvette containing 3 ml of distilled water. The optical transparency of the sample was measured as light transmittance (%) at 400-700 nm using a Hitachi LIH5300 UV-Vis spectrophotometer, with a quartz cuvette (3 cm optical path) (figure 1 and figure 2). The measurements were performed in the WL scan mode. Each sample was analysed in triplicate on silicone hydrogel lenses obtained as in example 1A); there were six concentrations of NPs-PGM used to produce the soaking solution in which to immerse the lenses (0.20 mg / mL, 0.30 mg / mL, 0.50 mg / mL, 0.75 mg / mL and 1.00 mg / mL)]. The concentrations of NPs-PGM loaded with FITC or atenolol to produce the soaking solution in which to immerse the lenses were such as to reach an FITC or atenolol concentration equal to 200 pg / mL. Considering that the ISO tolerance for light transmission must remain within ±5%, the incorporation of NPs loaded with drug / dye rendered the optical transparency of the lenses acceptable according to this criterion.

[0214] D) Study of the release of molecules to be loaded into the NPs deposited on the lenses

[0215] A release study is an experiment aimed at measuring the release kinetics of molecules (for example active ingredients, dyes (markers), pre / post-biotics).

[0216] Contact lenses coated with NPs loaded with molecules (for example active ingredients, dyes (markers), pre / post-biotics, as in examples 2A, 2B and 2C) were prepared.

[0217] Furthermore, an artificial tear saline solution (0.9% NaCI; 0.015% NaHCOs) was prepared.

[0218] The contact lenses were recovered from the preparation solution and dried with an absorbent cloth; then they were washed in a clean vial using 3 mL of artificial tear solution in order to eliminate any residues of unloaded nanoparticles. This washing step was repeated three times for each lens. Every washed lens was placed in a specific vial containing 2 mL of artificial tear solution and a magnetic anchor.

[0219] All the vials were placed on a magnetic shaking device at a temperature of 35°C, which corresponds to eye temperature.

[0220] Every hour, the lenses were taken out of the vials and dried with an absorbent cloth. 2 mL of solution were drawn from the vials and transferred into separate analytical vials. 2 mL of fresh artificial tear solution and the contact lens were again introduced into the initial vial. The vials were subsequently placed on a magnetic shaking plate at 35°C. These steps were repeated for every sample.

[0221] The releases gathered in the analytical vials were quantified using the instrument best suited to the compound encapsulated in the nanoparticles (for example, UV-Vis, fluorimeter, HPLC-MS). A curve was constructed based on the cumulative mass obtained from the various samples. Figure 3 shows the release velocity of a beta blocker drug (timolol), defined according to equation 3:

[0222] Equation 3 100 where rm and m2 are the mass of drug measured at the sampling times t1 and t2, respectively. From Figure 3 it may be observed that the release velocity is reduced by two orders of magnitude in the case of release occurring in the system of contact lenses loaded with nanoparticles containing the drug.

[0223] Example 6

[0224] Kinetics of mucin nanoparticle release from commercial contact lenses

[0225] In order to evaluate the release profile of the covalently cross-linked glycosylated mucin nanoparticles (NP) loaded onto commercial contact lenses, commercial lenses - both soft hydrogel-based lenses and soft silicone hydrogel-based lenses, as well as rigid gas-permeable lenses - were loaded by immersion in a suspension of NPs (250 pg / mL) for 24, 48 or 72 hours. After the loading step, the lenses were incubated in a saline solution at 4°C or 25°C (Figures 4A and 4B, respectively). The cumulative release of NPs was monitored over time up to 21 days.

[0226] At 25°C, all the tested conditions showed a progressive release depending on the time for loading the NPs onto the lenses, most of the release being concentrated in the first 72 hours. In particular, the lenses loaded for 48 and 72 hours showed comparable release profiles, reaching a cumulative release of 67% ± 7% on day 21. The release kinetics followed a characteristic trend: an initial burst phase (within the first 48-72 hours), followed by a plateau, indicative of a stabilisation of the release process. The lenses loaded for only 24 hours released up to 50% ± 7% of the total NPs under the same conditions, thus confirming the impact of the loading time on release efficiency. At 4°C, the overall release was markedly reduced throughout the duration of loading. The cumulative release reached only 9%, 10% and 18% for the lenses loaded for 24, 48 and 72 hours, respectively. Despite the smaller amounts, the release profile at 4°C reflected the one observed at 25°C, with most of the release occurring within the first 72 hours and minimal variations thereafter.

[0227] These results indicate that the NPs are released gradually by commercial contact lenses, with temperature and loading time playing a significant role in modulating the release efficiency. Higher temperatures and longer loading times favour a greater retention and subsequent release of NPs, supporting the feasibility of using contact lenses, preferably soft, as modulable release platforms for mucin-based nanoparticles.

[0228] In order to better evaluate the performance of the release system under physiologically relevant conditions, further analysis was conducted on the release of: (i) the free active ingredient (API, methylene blue), (ii) the nanoparticles loaded with the active ingredient (API in NPs), and (iii) the unloaded nanoparticles (NPs) from lenses immersed in simulated tear fluid (i.e. 0.9% NaCI and 0.015% NaHCO3) at 35°C (Figure 5).

[0229] The cumulative release of the free API (not loaded into nanoparticles) from lenses showed a rapid initial burst, reaching 82 ± 4% already at the first time point (30 min) and stabilising at 92 ± 4% in the subsequent ones. This behaviour is evidence of a low retention of the drug not encapsulated in NPs on the lens and suggests a rapid diffusion in the external medium.

[0230] In contrast, the release of empty NPs (NPs) was significantly slower, with a progressive increase from 5% to 22% over time. Similarly, the lenses loaded with NPs containing API (API in NPs) showed a profile of controlled release of the encapsulated active ingredient, with a cumulative release that stabilised at around 16% ± 7%. These data demonstrate that encapsulation in nanoparticles considerably slows down the release of the drug, thereby enabling a more prolonged administration compared to free API. Furthermore, the similarity between the release profiles of NPs including and not including API confirms that the presence of the methylene blue within the NPs does not significantly alter the overall dynamics of release of the nanocarrier from the lens.

[0231] Overall, these results support the ability of the mucin-based nanoparticles to modulate and prolong the release of the drug when incorporated into contact lenses, in particular under physiological conditions of temperature and tear fluid composition.

Claims

CLAIMS1. Contact lens comprising covalently cross-linked glycosylated mucin nanoparticles.

2. Contact lens according to claim 1 , characterised in that the covalently cross-linked glycosylated mucin nanoparticles are not covalently bound to the contact lens.

3. Contact lens according to any one of the preceding claims, wherein the mucin is a highly glycosylated protein, of natural or recombinant origin, characterised by the presence of one or more domains rich in serine and threonine residues which bind O-glycans containing saccharides selected from n-acetylgalactosamine, n-acetylglucosamine, fucose, galactose, sialic acids, and mannose, wherein the total serine and threonine content is greater than 10%, it is preferably comprised between 12% and 45%, and more preferably it is comprised between 15% and 36%, relative to the total quantity of amino acids of the protein.

4. Contact lens according to any one of the preceding claims, wherein the mucin is selected from mucin of animal origin, preferably pig gastric mucin, preferably type III, bovine submaxillary mucin, snail mucin, mucin obtained by recombinant methods, mucin encoded by a gene belonging to the MUC family, preferably MUC2, MUC5AC, MUC5B, MUC6, MUC7, or MUC19; lubricin, a TIM protein (T-cell immunoglobulin and mucin domain) and / or podocalyxin.

5. Contact lens according to any one of the preceding claims, wherein the contact lens is selected from a soft hydrogel-based contact lens, a soft silicone hydrogel-based contact lens and a rigid gas permeable contact lens.

6. Contact lens according to any one of the preceding claims, wherein the ratio between the mass of the nanoparticles and the mass of the lens is comprised between 1 :1000 and 1 :1 , preferably between 1 :100 and 1 :10, and even more preferably between 1 :90 and 1 :70.

7. Contact lens according to any one of the preceding claims, wherein the mass of covalently cross-linked glycosylated mucin nanoparticles is comprised between 0.005 and 5 mg, preferably between 0.01 mg and 1 mg and even more preferably between 0.05 and 0.5 mg8. Contact lens according to any one of the preceding claims, wherein covalently cross-linked glycosylated mucin nanoparticles reversibly coat the contact lens and / or are reversibly deposited on the contact lens.

9. Contact lens according to any one of the preceding claims, wherein the covalently cross-linked glycosylated mucin nanoparticles comprise at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

10. Method for preparing a contact lens according to any one of claims 1 to 9, comprising the following step: a) total or partial immersion of the contact lens in an aqueous suspension of covalently crosslinked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic.

11. Method for preparing a contact lens according to any one of claims 1 to 9, comprising the following step: a) depositing an aqueous suspension of covalently cross-linked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post-biotic, onto a contact lens by nebulisation.

12. Method according to claim 11 , wherein the contact lens is placed in close proximity to a nebuliser, preferably an airbrush sprayer, preferably at a distance of between 10 cm and 50 cm, more preferably between 15 and 25 cm.

13. Method according to any one of claims 11 to 12, wherein the duration of spraying is comprised between 1 minute and 10 minutes, more preferably between 2 and 5 minutes.

14. Method according to any one of claims 10-13, wherein the ratio between the mass of the nanoparticles and the mass of the lens is comprised between 1 :1000 and 1 :1 , preferably between 1 :100 and 1 :10, and even more preferably between 1 :90 and 1 :70.

15. Contact lens according to any one of claims 1 to 8, for use in treating dry eye.

16. Use of the contact lens according to any one of claims 1 to 9, for controlled release of at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post- biotic.

17. Use of covalently cross-linked glycosylated mucin nanoparticles, optionally comprising at least one compound selected from an active ingredient, a marker, a biomolecule and / or a pre / post- biotic, for coating a contact lens, preferably selected from a hydrogel-based soft contact lens, a silicone hydrogel-based soft contact lens and a rigid gas-permeable contact lens.

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