WO2019238610A1

WO2019238610A1 - Keratin nanofibers as delivery vehicles of active ingredients, methods for the production and uses thereof

Info

Publication number: WO2019238610A1
Application number: PCT/EP2019/065079
Authority: WO
Inventors: Greta Varchi; Annalisa ALUIGI; Giovanna Sotgiu
Original assignee: Kerline S.R.L.
Priority date: 2018-06-12
Filing date: 2019-06-10
Publication date: 2019-12-19
Also published as: EP3806816A1; WO2019238610A8

Abstract

Nanofibers comprising a water-soluble keratin polypeptide, at least one active ingredient and optionally at least one supporting polymer, wherein the nanofiber is obtained by electrospinning an aqueous solution comprising the keratin polypeptide, the active ingredient and optionally a supporting polymer, such as for example polycaprolactone, polylactic acid, polyglycolic acid and poly(lactic-co-glycolic acid); a method for preparing such nanofibers, and pharmaceutical and cosmetic formulations containing them are also described.

Description

Title: KERATIN NANOFIBERS AS DELIVERY VEHICLES OF ACTIVE INGREDIENTS, METHODS FOR THE PRODUCTION AND USES THEREOF

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to the field of the pharmaceutical, biomedical and cosmetic industry.

In particular, the present invention relates to water-soluble keratin nanofibers which incorporate active ingredients to be delivered to cells and tissues in order to improve the properties of such active ingredients, such as for example: bioavailability, local and controlled diffusion of the active ingredient.

The present invention also relates to the methods of preparation of the aforementioned keratin nanofibers incorporating active ingredients.

BACKGROUD OF THE INVENTION

Recently, the development of polymeric nanofibers obtained by electrospinning as topical and transdermal drug delivery systems has shown great potential [ 1 ] .

It is known that electrospinning is a process by which a polymer in a solution can be spun into ultra-thin fibers in the presence of a high potential electric field. In particular, this technique allows to obtain polymeric fibers with a variable diameter comprised between 3 nm and 4 pm; thank to this feature, the nanofibers obtained by electrospinning have several advantages, including for example a high surface-to-volume ratio, porosity and a diameter variable depending on the electrospinning conditions.

These properties make these nanofibers suitable for different fields, including for example: environmental engineering, biotechnology, medicine and cosmetics.

An application of growing interest concerns a delivery system of molecules loaded on said nanofibers , wherein the selection of the polymer necessary for the nanofibers production by electrospinning is of utmost importance; in fact, the interactions that may be established between the active ingredients, the polymer to be electrospun and the solvent in which the electrospinning occurs, may alter the correct formation of nanofibers, their morphologic, mechanical and chemical-physical properties.

It is known that synthetic polyamide- or polyurethane-based nanofibers can be obtained by electrospinning, showing interesting elastic properties and mechanical strength.

However, in recent years, research interest has shifted to natural polymers.

Among natural polymers, proteins such as, for example, collagen, gelatin, elastin and fibrinogen are particularly preferred, because their structural characteristics facilitate the loading of molecules onto the nanofibers obtained from said materials. Moreover, these proteins can load and deliver both positively and negatively charged molecules as well as hydrophilic and / or hydrophobic molecules.

Compared to commercially available proteins (e.g. collagen), keratin is a widely distributed biopolymer, being the major component of hair, hair, wool, feathers, skin, nails and has excellent biological properties, such as, for example, biocompatibility, biodegradability, and an intrinsic anti inflammatory and tissue regeneration effect [2].

Moreover, the primary structure of keratin is known to comprise amino acid sequences that are able to interact and bind specific cell receptors, including for example the leucine - aspartic acid - valine (LDV) sequence and the glutamic acid - aspartic acid - serine sequence (EDS), which can promote cell adhesion and proliferation [3].

The transformation of keratin polypeptides into nanofibers by electrospinning is also known.

In particular, keratin can be electrospun alone or in a mixture with another polymer (for example, polyethylene oxide, polyvinyl alcohol, polycaprolactone, polylactic acid), providing keratin-based nanofibers which, being arranged in membranes, can find application in several technological fields, including the biomedical field, for example to promote the wound healing process.

Moreover, keratin arranged in nanofibers guarantees tissue transpiration, cell adhesion and regeneration, due to its specific amino acid sequences.

The scientific article "Electrospinning of Keratin / Poly (ethylene oxide)", by Aluigi et al. (Journal of Applied Polymer Science, Vol. 104, 863-870 (2007)) describes the production and characterization of keratin and PEO nanofibers in a 50:50 ratio (by weight), produced by electrospinning in aqueous solution, under several operating conditions; the nanofibers have been studied with scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) and the results have been compared with those obtained from thin films produced by casting from the same solutions with the aim of investigating the influence of the production processes on the structural arrangement of these materials.

The scientific paper "Fabrication and Characterization of Electrospun PCL- MgO-Keratin-Based Composite Nanofibers for Biomedical Applications" by Maame AD Boakye et al., (Materials 2015, 8, 4080-4095; doi: 10.3390 / ma8074080) describes electrospun nanofibers of poly (e-caprolactone) / keratin loaded with magnesium oxide (PCL-K / MgO), whose morphology was analyzed using SEM technique; the chemical composition and surface chemistry were studied by FTIR and the mechanical properties of the nanofibers were evaluated for the "tensile loading". Furthermore, a study was carried out on the release of magnesium oxide (involved in various cellular processes, such as cellular respiration, protein synthesis, membrane integrity), in order to quantify the amount of magnesium delivered into the environment by the aforementioned nanofibers.

The publication "Tailoring Properties and Functionalities of T1O2 and Ag nanoparticles involved in surfaces engineering processes" by Ortelli S. (Dissertation thesis, DOI 10.6092 / unibo / amsdottorato / 6968) describes keratin/ polyethylene oxide (PEO) nanofibers loaded with nanoparticles of silver and titanium dioxide whose properties are mainly antibacterial and photocatalytic . In particular, the incorporation of these inorganic nanoparticles into keratin / PEO nanofibers resulted in the formation of nano-reactive membranes for the removal of different classes of pollutants from water and air.

The scientific paper“Dual-layered 3D nanofibrous matrix incorporated with dual drugs and their synergetic effect on accelerating wound healing through growth factor regulation” by Sivakumar Singaravelu et al., (Materials Science and Engineering C 76 (2017) 37-49) describes a spongy 3D matrix consisting of a double layer of nanofibers obtained by electrospinning and their use in tissue engineering; in particular, one layer consisted of poly-(3-hydroxybutyric acid) / gelatin nanofibers loaded with curcumin, the other layer consisted of keratin / fibrin / gelatin nanofibers loaded with mupirocin. In particular, the properties of the 3D matrix was evaluated on wound healing in vivo , taking into account the changes in expression levels of VEGF, EFG and TGF-b in Wister albino rats

Therefore, all the aforementioned documents teach that keratin nanofibers can be electrospun in an aqueous environment, mixed with a polymer of synthetic origin.

Some of the previous documents also indicate that these nanofibers can be loaded with different products, such as nanoparticles, salts or natural occurring molecules.

However, none of the aforementioned documents provides a nanofiber made of an electrospun natural polymer, which is able to load and deliver an effective amount of at least one active ingredient.

Zhang J.,“Keratin composite nanofibrous anti-tumor drug delivery system” (The Hong Kong Polytechnic University-Dissertation, Hong Kong) discloses keratin/PLLA nanofibers loaded with 5-fluorouracil and/or the antimicrobial peptide attacin2 obtained by electrospinning a 5-FU/keratin suspension blended with PLLA organic solution (90% chloroform and 10% DMF).

CN 104 018 247 discloses a polyurethane/ keratin composite nanofiber material obtained by electrospinning a hexafluoroisopropanol (HFIP) solution comprising polyure thane and keratin.

CN 103 993 426 discloses a polyethylene terephthalate-keratin composite nanofiber material obtained by electrospinning a solution comprising polyethylene terephthalate and keratin, the solvent being one or more of hexafluoroisopropanol, N,N-dimethylformamide (DMF), N,N- dimethylacetamide (DMA), N-methyl-pyrrolidone, dichloromethane, chloroform, tetrahydrofuran (THF) and ethyl chloride, preferably hexafluoroisopropanol.

CN 104 005 179 discloses polycaprolactone-keratin nanofibers obtained by electrospinning a solution comprising polycaprolactone and keratin; the solvent being HFIP or formic acid. ay et al.,“Effects of solvents mixtures on the morphology of electrospun thermoplastic polyure thane nanofibers” (Teksil ve Konfeksiyon 25(1), 1 March 2015, pages 38-46) teaches that solvents used in the electrospinning solution/ suspension have an effect on the morphology of the resulting nanofibers, such as diameter and porosity.

None of the above-mentioned documents discloses a keratin nanofiber obtained by electrospinning in an aqueous solution.

Therefore, the objective technical problem on which the present invention is based is that of providing electrospun nanofibers of a natural polymer, preferably a keratin polypeptide, which are able to load a predetermined amount of at least a cosmetic, phytotherapeutic or pharmaceutical, lipophilic or hydrophilic active ingredient, while keeping unchanged their mechanical and elastic properties.

SUMMARY OF THE INVENTION

Such problem was solved, according to the invention, by a nanofiber comprising a water-soluble keratin polypeptide, at least one active ingredient and optionally a supporting polymer, said nanofiber being obtained by electrospinning an aqueous solution comprising said keratin polypeptide, said at least one active ingredient, and optionally a supporting polymer.

The above-mentioned aqueous solution can further comprise a surfactant. The surfactant can be selected from anionic surfactants, cationic surfactants, non-ionic surfactants or mixtures thereof.

Preferably, the supporting polymer is selected from the group consisting of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyure thane, hyaluronic acid, polyhydroxy alkanoates (PHAs), poly butylene succinate (PBS), cellulose and derivatives thereof (methyl cellulose, hydroxymethylcellulose), starch, chitosan, fibroin, collagen, elastin, polyethylene glycol, polypropylene glycol, polymethylmethacrylate, prolamin, zein, pectin, casein, gluten, soy proteins, microalgae proteins, sericin, resilin, chitin, polyamides, polyethylene, polyethylene- terephthalate, poly butylene terephthalate, polypropylene, polyvinylidene fluoride, polycarbonate, acrylonitrile-butadiene-styrene, polyvinyl chloride, polyvinyl fluoride and polyoxymethylene.

Preferably, the keratin polypeptide is selected from the group consisting of wild-type keratin polypeptides, chemically modified keratin polypeptides and mixtures thereof.

Preferably, the keratin polypeptide has a molecular weight comprised between 150 kDa and 3.5 kDa, preferably 45 kDa.

Preferably, the nanofiber has a diameter comprised between 50 nm and 1000 nm, preferably comprised between 100 nm and 900 nm.

Preferably, the at least one active ingredient is in an amount between 0.01% and 20% by weight of the weight of the nanofiber.

Preferably, the nanofiber as defined above comprises, in percentage by weight of the total weight of the nanofiber, 0.1-99% of keratin polypeptide, 0.01-20% of said at least one active ingredient and 5-80% of said supporting polymer.

Preferably, the at least one active ingredient is selected from the group consisting of therapeutic agents, biologically active molecules, diagnostic agents, nutraceuticals, cosmetic ingredients, phytotherapeutic agents, natural products, dyes, cosmeceuticals, vitamins and trace elements.

Preferably, the therapeutic agent, biologically active molecule or diagnostic agent are selected among the group consisting of anti-inflammatory agents, anticancer drugs, antimicrobial agents, analgesics, hormones, local and general anesthetic agents, bone regeneration agents, osteoinductive agents, osteoconductive agents, biomaterials for osteogenesis, demineralized bone matrix, collagen, tricalcium phosphate, hydroxyapatite, coraline bone substitutes, bioactive glasses, glass ionomers, antianginals, anti- arrhythmic drugs, antibacterial and antiprotozoal agents, anticoagulants, antidepressants, anti-diabetic drugs, anti-epileptic drugs, antifungal agents, antihistamines, anti-hypertensive drugs , anti-muscarinic agents, antineoplastic agents, anti-migraine drugs, antiparasitic agents, anti- Parkinson drugs, antipsychotic, hypnotic and sedative agents, anti-stroke agents, anti-thrombotic agents, antitussives, antivirals, beta blockers, calcium channel blockers, cardiac inotropic agents, contraceptive agents, corticosteroids, dermatological agents, diuretics, gastro-intestinal agents, haemostatics , local anesthetics, opioid analgesics, para- sympathomimetics, peptides, steroids, stimulating agents, vasodilators, preferably anti-inflammatory drugs, drugs for dermatological diseases, antifungal for dermatological use, emollients and protectives for the treatment of wounds and ulcerations, antipruritics, including antihistamines and anesthetics, anti-psoriatic agents, antibiotics and chemotherapeutics for dermatological use, corticosteroids, antiseptics and disinfectants, antiacne agents.

In one aspect thereof, the invention also describes a pharmaceutical composition comprising the above-mentioned nanofibers and a pharmaceutically acceptable carrier.

In another aspect thereof, the invention describes a pharmaceutical formulation comprising the aforementioned composition for administration by enteral, particularly oral and rectal, route, parenteral, particularly subcutaneous and intradermal, route, transcutaneous route, sublingual and transmucosal, particularly ocular, nasal, vaginal and urethral route.

Preferably, the pharmaceutical formulation is for use in the treatment of dermatological diseases, neoplastic diseases, neurodegenerative diseases, metabolic diseases, cardiovascular diseases and for bone reconstruction.

In a further aspect thereof, the invention also describes a cosmetic formulation comprising nanofibers in which the active ingredient is a cosmetic ingredient, a nutraceutical, a phytotherapeutic agent, a natural product, a dye, a cosmeceutical, a vitamin, a trace element and a cosmetically acceptable vehicle. Preferably, the nanofibers of the aforementioned pharmaceutical formulation for transdermal or topical application form a membrane.

Preferably, the nanofibers of the aforementioned cosmetic formulation for transdermal or topical application form a membrane.

Preferably, the aforementioned cosmetic formulation is for the treatment of keratinous substrates, such as skin, nails and hair.

Preferably, the aforementioned cosmetic formulation is for treating the skin aging signs and / or improving the appearance of the skin.

In another aspect thereof, the present invention relates to a method for producing a nanofiber as described above, comprising the steps of:

- dissolving in water the aforementioned water-soluble keratin polypeptide, the at least one active ingredient and optionally the supporting polymer;

- electrospinning the thus obtained solution.

In the above-mentioned step of dissolving in water the aforementioned water-soluble keratin polypeptide, the at least one active ingredient and optionally the supporting polymer, a surfactant can also be dissolved in water.

The surfactant can be selected among anionic, cationic and non-ionic surfactants; examples of preferred surfactants that can be used in the present invention are the sorbitan esters, in particular those known as Spans^® and the polysorbates known as Tweens^® (e.g. Tween^® 20), and other non-ionic surfactants such as e.g. Triton X- 100.

The present invention is based on the finding that incorporation of one or more active ingredients (up to four ingredients), such as for example a drug, a nutraceutical, a cosmetic, an antibacterial compound, a local anesthetic, a food supplement, a dye, an organic or inorganic compound, takes place in an effective and stable manner into nanofibers obtained from the electrospinning of water-soluble keratin polypeptides.

Advantageously, the keratin nanofibers according to the present invention improve the bioavailability and efficacy of the loaded active ingredients, while reducing their tissue toxicity. Furthermore, the loading of an active ingredient into said water-soluble keratin nanofibers can promote tissue penetrability.

According to the present invention, the keratin nanofibers can also comprise two or more active ingredients with different solubility degrees in water or in solvents such as formic acid, hexafluoroisopropanol, dimethylformamide, i.e. being lipophilic or hydrophilic, positively charged, negative charged or neutral.

The possibility of loading such different active ingredients allows the freedom to choose the most suitable active ingredients for the treatment of a specific pathological condition, irrespective of their water solubility, which is a crucial parameter to guarantee the correct delivery of active ingredients to the target organ or tissue.

Another important advantage of the keratin nanofibers of the present invention, obtained by electrospinning, is the high area/ volume ratio and their highly porous structure, which promotes cell growth and controlled delivery of the active ingredient.

According to another aspect of the invention, the water-soluble keratin can be chemically modified to improve its selectivity for specific cells and tissues, or to improve its physico-chemical properties, while maintaining its solubility in water.

A further advantage of the present invention is that the keratin polypeptide described herein is not significantly hydrolyzed during its extraction, thus it has a molecular weight comprised between 150 and 3.5 kDa, preferably of 45 kDa. The characteristics of these keratin polypeptides (molecular weight, primary and secondary structure) are found into the nanofibers, favoring tissue regeneration due to the maintenance of the amino acid sequence.

According to the present invention, the electrospinning of keratin polypeptide nanofibers takes place in water, thanks to the use of water- soluble keratin polypeptides for the formation of the nanofibers; this results in considerable advantages in terms of safety and environmental impact, without compromising the efficiency of the procedure, which becomes particularly relevant when electrospinning takes place at industrial scale.

Advantageously, the presence of said further supporting polymer allows to obtain nanofibers with mechanical properties adequate and modular depending on the application, with porosity suitable for the regeneration of different types of cells (fibroblasts, osteoblasts, neuroblasts), for applications in tissue engineering field.

Advantageously, the presence of said further supporting polymer also allows to obtain nanofibers of adequate and modular dimensions allowing to obtain a controlled delivery of active ingredient, suitable for therapeutic and/or cosmetic treatment.

A "nanofibe , as used herein refers to a nanofiber obtained by electrospinning of a keratin polypeptide, optionally mixed with another polymer.

A“polypeptide” as used herein refers to a consecutive series of 2 or more amino acids and as used herein, a “polypeptide” comprises the term “protein”. Therefore, in some embodiments, the polypeptide comprises a consecutive series of at least 10 to 800 amino acids.

The expression “modified keratin” as used herein refers to a keratin polypeptide including one or more additional amino acid residues at the C- terminus or N-terminus of the consecutive sequence of amino acids of the keratin polypeptide.

In particular, the modified keratin polypeptide has at least 50% to 99% sequence homology to a known wild type keratin protein; the keratin polypeptide may include a chemical functionalization with organic compounds at the N-terminus or hydroxyl, amino and thiol groups of amino acid side chains.

The expressions “highly water-soluble keratin” and “highly hydrosoluble keratin” are used herein as synonyms and are meant to indicate a keratin having a solubility in water or in a buffered isotonic solution of at least 150 mg/mL at a temperature of 25°C.

The expression "supporting poly me as used herein refers to a polymer selected from natural and synthetic polymers such as, but not limited to, polycaprolactone, polylactic acid, polyglycolic acid, poly lactic-co-glycolic acid, poly-urethane, hyaluronic acid , poly-hydroxy alkanoates (PHAs), poly-butylene succinate (PBS), cellulose and its derivatives (methylcellulose, hydroxymethylcellulose), starch, chitosan, fibroin, collagen, elastin, poly-ethylene glycol, polypropylene glycol, poly-methyl methacrylate, prolamine, zein, pectin, casein, gluten, soy protein, sericin, resilin, chitin, polyamides, polyethylene, polyethylene-terephthalate, poly butylene terephthalate, polypropylene, polyvinylidene fluoride, polycarbonate, acrylonitrile-butadiene-styrene polyvinyl chloride, polyvinyl fluoride and polyoxymethylene.

Preferably this supporting polymer is selected from polycaprolactone, polylactic acid and hyaluronic acid.

The term "electrospinning", as used herein, refers to a technique that allows, in the polymer industry, to obtain fibers with a small diameter (less than 1000 nm), starting from a keratin polypeptide in water or aqueous solution, according to the protocol described in [4] .

The expressions "electrospun keratin polypeptide " or "electrospun keratin " are used here as synonyms and indicate that the keratin polypeptide has been subjected to the electrospinning technique as defined above.

The expression " nanofiber loaded with at least one active ingredient , as used herein, is meant an electrospun keratin that incorporates at least one active ingredient in a variable percentage ranging from 0.01 to 20% by weight of nanofiber.

A "drug" or " active ingredient as used herein refers to any therapeutic or diagnostic agent, or biologically active molecule. The drugs include agents that are used in the treatment and prevention of different diseases, such as, but not limited to, neoplastic diseases, infectious diseases, dermatological diseases, neurodegenerative diseases, metabolic diseases, cardiovascular diseases. Furthermore, they can be bone regenerators, osteoinductors, osteoconductors and biomaterials for osteogenesis. The drug can be a small organic or inorganic molecule, a peptide, a polypeptide, a protein, a carbohydrate, an antibody, an antibody fragment, DNA, RNA, miRNA, siRNA or a combination thereof. Non-limiting examples of therapeutic agents include an anti-inflammatory agent, an antimicrobial agent, a hormone, an anticancer agent, an agent for the treatment of degenerative diseases, an anesthetic agent, an agent for the treatment of dermatological diseases, an agent for the treatment of metabolic pathologies, an antipruritic agent, a modulator of the genital system, an antirheumatic agent, an antiparasitic agent.

A "cosmetic ingredient , as used herein, refers to any chemical compound used in cosmetic products.

A " nutraceutical ingredient , as used herein, refers to nutrients, dietary supplements and herbal products to be included in specific diets or processed foods such as, for example, vegetable / animal oils, vitamins, cholesterol, creatine, amino acids, mineral salts, beta-carotene, flavonoids, vegetable or yeast extracts, hyaluronic acid, inositol, herbs, and all the other components listed in the EU Regulation n. 432/2012 and encompassing the definition of Dietary Supplement Health and Education Act of 1994.

A " natural product or " natural product extract as used herein means an organic or inorganic compound obtainable from nature environment such as, but not limited to, a product generally recognized as safe (GRAS), antibiotics, fats, oils, petrochemical derivatives, resins, toxins, foods, metals and natural metal compounds, minerals, rocks, soils, nucleic acids, organisms (including bacteria, multicellular plants and animals), proteins, peptides, phytochemicals and phytotherapeutic compounds.

A " cosmeceutical ingredient , as used herein, refers to all the ingredients that can be chosen from those specified in the database "Cosing" of the European Union

(https : / / ec. europa. eu / growth / sectors / cosmetics / cosing_en) , which includes the "Inventory of Cosmetic Ingredients", and which present benefits for the body similar to those provided by a drug, such as vitamins, alpha- and beta-hydroxy acids, trace elements, lipoic acid, di-methyl amino ethanol, glycolic acid, salicylic acid and hyaluronic acid.

A“dye” as used herein references to any organic or inorganic compound natural or synthetic to be used for colouring textiles, foods, hair, polymers, cells, tissues, etc, such as, for example, porphyrins, phtalocyanines, hair color dyes, dyes for coloring fabrics, organic and inorganic pigments for fabrics and polymer fabrics.

The expression“dermatological disease” as used herein refers to any skin disease which may have visible or not-visible consequences, including: cell differentiation and proliferation diseases, keratinisation disorders, inflammatory and allergic disorders, sebaceous disorders, dermis diseases, malignant or not malignant epidermal proliferations, skin diseases caused by UV radiation, disease associated with aging, skin cicatrization diseases, scleroderma, miastenia gravis, organ rejection, endotoxin shock, sepsis, psoriasis, eczema, atopic dermatitis, bullous dermatitis, Louis-Bar’s syndrome, Cowden’s syndrome, dyskeratosis congenital, Rubinstein- Taybi’s syndrome, Werner syndrome, xeroderma pigmentosum, multiple sclerosis, autoimmune thyroiditis, uveitis, systemic lupus erythematosus, Addison's disease, autoimmune polyglandular syndrome and Graves' disease.

The term "neoplastic diseases" as used herein refers to diseases that include, but are not limited to, tumors and carcinomas such as, pancreatic, mammary, male breast, colon, colorectal, prostatic, gastrointestinal stromal, adrenal, ovarian tumors, skin melanoma, non-skin melanoma, non-melanoma skin, mouth , eye, nasal cavity and paranasal sinuses, lung, penile , bronchial, heart, uterine body, cervix , esophageal, liver, metastatic hepatic, lymphomas (Hodgkin, non-Hodgkin, cutaneous , pediatric lymphomas), larynx, pharynx, malignant mesothelioma, leukemia (hairy cells, chronic lymphatic, acute lymphoblastic, acute myeloid, chronic myeloid, pediatric leukemia), multiple myeloma, sarcomas, gliomas, kidney, testicular cancer, thyroid, soft tissue sarcoma, bone, Ewing sarcoma, Kaposi sarcoma, extraskeletal Ewing sarcoma, chondrosarcoma, osteosarcoma, metastatic bone, choriocarcinoma, pineal gland, salivary gland tumors, hypophysis cancer, primitive neuroectodermal tumor, multiple endocrine neoplasia type 1 , Multiple Endocrine Neoplasia Type 2, bladder, pelvic organs, ureteral, oral cavity, anal, vulvar, vaginal, spleen, brain tumors, embryonal tumors, gall-bladder, bile duct, cancer cachexia, neuroblastoma, pediatric neuroblastoma, neuroendocrine, pediatric tumors and Myeloproliferative neoplasms.

The term " neurodegenerative disease" as used herein refers to diseases that include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Lewy body dementia, corea-acanthocytosis, primary lateral sclerosis, eye diseases (ocular neuritis), chemotherapy-induced neuropathies (eg, by vincristine, paclitaxel, bortezomib), neuropathies induced by diabetes and Friedreich's ataxia.

The term " metabolic disease" as used herein refers to diseases that include, but are not limited to, hereditary obesity, dietary obesity, hormonal obesity and obesity related to drug administration, hypertension, high cholesterol, dyslipidemia, type 2 diabetes and / or their complications, insulin resistance, pre-diabetic state, glucose intolerance, hyperinsulinemia, coronary artery disease, angina pectoris, congestive heart failure, stroke, gallstones, cholecystitis and cholelithiasis, gout, osteoarthritis, obstructive sleep apnea and respiratory problems, some types of cancer (such as endometrium, breast, prostate and colon), pregnancy complications, poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation), bladder control problems (such as stress incontinence); uric acid kidney stones; psychological disorders (such as depression, eating disorders, distorted body image and low self-esteem), dysregulation of appetite and / or satiety, excess adipogenesis or differentiation of fat cells, reduced sensitivity to insulin and / or dysregulation of insulin levels, dysregulation of a person's weight and diseases and disorders related to a subject's weight loss.

The term "cardiovascular disease" as used herein refers to diseases that include, but are not limited to, cardiomyopathy or myocarditis, idiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholic cardiomyopathy, drug-induced cardiomyopathy, ischemic heart disease, hypertensive cardiomyopathy, atheromatosis of the major blood vessels (examples: aorta, coronary arteries, carotid arteries, cerebrovascular arteries, renal arteries, iliac arteries, femoral arteries, popliteal arteries), platelet aggregation (examples: retinal arterioles, glomerular arterioles, vasa nervorum, cardiac arterioles, capillaries connected to 'eye, kidney, heart, and the central and peripheral nervous system), restenosis, for example following coronary intervention, disorders related to an abnormal high-density and low-density cholesterol level in an individual's plasma.

The term "bone reconstruction" as used here refers to prostheses that include, but are not limited to, dental prostheses, neuroprostheses, orthopedic prostheses and breast implants.

The expression " treating keratin substrates" as used herein refers to any treatment aimed at preserving or restoring the healthy functioning of the skin and / or hair and / or nails or any treatment that provides means to preserve or improve their appearance and / or structure. Examples of such treatments include: skin strengthening , wrinkle reduction , moisturizing, protection from any kind of aggression, in particular, protection from sun radiations and aging indicators.

The expression "signs of aging " as used here includes all the changes regarding the appearance of the skin due to aging and photo-aging. Examples of these changes include wrinkles and thin lines, floss skin, thin skin, loss of skin elasticity and / or tone, opaque skin. It also includes internal modifications that do not directly affect external appearance changes. An example of such internal modifications is the degradation that occurs internally to the skin due to repeated exposure to UV radiation.

The expression "improvement of the appearance of the skin" as used herein includes all the phenomena that may result in a visual improvement of the skin appearance. Examples of such phenomena lead to a more beautiful, firm and smooth skin, diminishing or removing all the small imperfections, such as the attenuation of skin“orange peel effect”.

The expression " cosmetic formulation" , as used herein refers to a product having moisturizing and preserving properties and which is able to treat keratin substrates, signs of aging, skin damages due to exposure to environmental agents, thus improving the appearance of the skin.

The terms "external aggression" or " environmental aggression" as used herein refers to aggressions that may be of chemical, physical, biological or thermal origin.

The present invention also includes pharmaceutical compositions containing said hydrosoluble keratin nanofibers and conventional, non toxic and pharmaceutically acceptable carriers, excipients, adjuvants, rheology modifiers, surfactants and vehicle. An “excipienf as used herein references to any substance formulated alongside the active ingredient of a medication, included for the purpose of long-term stabilization, bulking up solid formulations that contain potent active ingredients, or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life.

The pharmaceutical compositions according to the present invention can be administered with any available and effective administration system, including, but not limited to: enteral route, including oral and rectal, transdermal dermal, sublingual and transmucosal routes including ocular, nasal, vaginal and urethral.

The solid dosage forms o by oral route include, for example, capsules, tablets, optionally obtained by functional coating or encapsulation, powders, granules and gels. These dosage forms normally also include additional substances different from inert diluents, such as, for example, lubricating agents like magnesium stearate.

The pharmaceutical preparations according to the present invention may be produced using conventional pharmaceutical techniques, as described in the various pharmacopoeias or handbooks of the field such as, for example, "Remington's Pharmaceutical Sciences Handbook", Mack Publishing, New York, 18th Ed., 1990.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present invention and are included to further demonstrate certain aspects of the invention. The invention may be better clarified if referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

Figure 1 shows keratin / PEO nanofibers in 80/20 ratio, loaded with diclofenac sodium salt (3% by weight of the keratin weight). Figure 2 presents a graph of diclofenac delivery from keratin / PEO nanofiber membranes in 80/20 ratio according to the present invention.

Figure 3 shows a keratin / PEO nanofiber membrane in 80/20 ratio comprising curcumin (3% by weight of the keratin weight) as an active ingredient, according to the present invention.

Figure 4 shows the application of a keratin / PEO nanofiber membrane in 80/20 ratio and its adhesion ability on a keratin substrate, according to the present invention.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is based on the finding that keratin nanofibers can be used for the incorporation and delivery of at least one or more hydrophobic active ingredients or of at least one or more hydrophobic and hydrophilic active ingredients.

Furthermore, the keratin nanofibers, as compared to other polypeptides and proteins, allow the stably incorporate high amount of a selected active ingredient, due to their high porosity and high surface /volume ratio.

Interestingly, keratin included into nanofibers has a therapeutic function itself since, as known, this protein is involved in tissue repair following, for example, burns and wounds.

Consequently, the keratin nanofibers of the present invention, loaded with at least one active ingredient, show multiple biological functions: those determined by the activity of the active ingredients loaded on the nanofibers, and those determined by the activity of keratin of which the nanofibers are made.

The aforementioned keratin nanofibers are obtained by electrospinning in water.

Briefly, electrospinning is a process of spinning a polymer in solution, driven by an electric force. This process is quite simple and consists of a pump, a syringe, a spinneret, a voltage source and a collector.

The spinneret is connected to a syringe containing a keratin polypeptide in water, optionally mixed with a supporting polymer.

Thanks to the pump, the polymer passes through the spinneret with a constant and controllable flow. When a high potential difference is applied between the syringe capillary and the collector, a drop of extruded polymer is formed at the spinneret tip.

As the voltage difference increases, the drop is subjected to a repulsive force between its surface charges and the Coulomb force exerted by the external electric field. Under the action of these forces, the drop is then distorted into a conical shape, known as "Taylor cone". When the electric field exceeds a threshold value, the electrostatic forces prevail over the surface tension of the polymer drop causing the expulsion of a liquid jet from the spinneret tip towards the collector. While traveling the distance towards the collector, the solvent evaporates, thus increasing the surface charge of the jet which, in turn, induces instability of the jet. This instability causes a process of stretching and "whipping motion", thus leading to the formation of a long and extremely thin filament. Attracted by the grounded collector, the charged nanofiber stars to deposit on the collector as a non-woven fabric, with random orientation.

According to an embodiment of the present invention, the water-soluble keratin polypeptide is mixed with a supporting polymer as defined above.

The present invention foresees that keratin nanofibers (or keratin in mixture with further said supporting polymer) are loaded with at least one lipophilic or hydrophilic, positively charged, negatively charged or neutral active ingredient (at most four active ingredients).

Being suitable for delivering active ingredients with different chemical/ physical characteristics, the nanofibers of the present invention are highly suitable for the pharmaceutical and cosmetic fields.

In an embodiment, the aforementioned nanofibers are arranged to form a membrane, whose characteristics can be optimized depending on its use; for example, the thickness of the nanofiber membrane can be adjusted by controlling the fineness and compactness as well as the porosity of the nanofibers. Thanks to the advantageous properties of the water-soluble keratin nanofibers, the thus-obtained membrane has excellent adhesion properties and is therefore effective in ensuring the delivery of the active ingredient loaded into the nanofibers as well as favoring the regeneration of the epidermal layer due to the presence of the keratin components.

The invention will now be described with the following examples, intended to better understand its contents, without limiting its scope.

EXAMPLES

Example 1

Keratin was extracted from raw wool by sulphitolysis reaction. Briefly, a fiber sample, withdrawn from a combed sliver and cleaned by Soxhlet extraction with petroleum ether, was washed with distilled water and dried at 21 °C and 60% relative humidity overnight. Afterward, cleaned fibers (5 g) were cut into snippets and dispersed in 100 mL of aqueous solution containing urea (8 M), sodium metabisulphite (0.5 M) and sodium dodecyl sulfate (SDS, 0.1 M), under mechanical shaking at 65 °C overnight. The mixture was filtered with a vacuum filter (10- 16 mih cut-off), dialyzed against distilled water using a cellulose tube (molecular weight cut-off 12- 14 kDa) for 3 days at room temperature, changing the distilled water four times a day. The resulting aqueous solution was freeze-dried in order to obtain pristine water-soluble keratin powder.

Example 2

Keratin / polyethylene oxide (PEO) nanofibers loaded with an active ingredient, i.e. diclofenac, was prepared according to the following protocol.

100 mg of pure water-soluble keratin (obtained according to Example 1) and polyethylene oxide were mixed in 80/20 ratio and subsequently dissolved in deionized water. Diclofenac (sodium salt) was added to the solution at a concentration of 3% by weight of the weight of keratin.

The solution was electrospun using 25 kV voltage , 0.05 mL/min flow rate , where the electrode distance was 15 cm.

Keratin / PEO nanofibers with an average diameter of about 290 nm were obtained, as shown in Figure 1.

Example 3

The diclofenac delivery from a keratin / PEO nanofiber membrane obtained according to Example 2 was evaluated by inserting weighted nanofiber samples (about 7 mg) in cellulose acetate membranes (12- 14 kDa cut-off) and by inserting them into vials containing 1.8 ml PBS at pH 7.4. They were closed in a stove and placed on a shaker and kept at 37 ° C, in order to simulate the physiological conditions. At certain time intervals, 300 mΐ of the medium were taken from each sample, followed by the addition of the same amount of PBS at pH 7.4. The diclofenac delivered over time was calculated by measuring the absorbance at 267 nm with UV-vis spectrophotometer, using the corresponding deliveries from membranes of non-loaded nanofibers as the reference sample.

Figure 2 shows the delivery of diclofenac from the membrane of the keratin nanofibers of the present invention.

As shown, there is a rapid delivery of the active ingredient, greater than 60% during the first 24 hours, with a total delivery of the active ingredient after 168 hours.

Example 4

A keratin / polyethylene oxide (PEO) nanofiber was obtained as described in Example 2. Curcumin was dispersed in the solution at a concentration of 3% by weight of the weight of keratin.

The suspension was electrospun using 25 kV voltage, 0.05 mL / min flow rate , where the electrode distance was 15 cm.

Example 5

The keratin / polyethylene oxide (PEO) nanofiber membrane prepared according to Example 2 was applied to a wet skin substrate (Figure 4), exploiting the adhesive properties of the membrane itself in the presence of a small percentage of water.

References 1. Waters M., VandeVord P., Van Dyke M.,“Keratin biomaterials augment anti-inflammatory macrophage phenotype in vitro.”, Acta Biomater. 2018 Jan 15;66:213-223. doi: 10.1016/j.actbio.2017.10.042. Epub 2017 Oct 28.

2. Jillian G. Rouse and Mark E. Van Dyke, “A Review of Keratin-Based Biomaterials for Biomedical Applications”, Materials 2010, 3(2), 999- 1014.

3. Yu B, Tai HC, Xue W, Lee LJ, Lee RJ. Receptor-targeted nanocarriers for therapeutic delivery to cancer. Mol Membr Biol. 2010;27:286-298.

4. Reneker DH, Chun I. Nanometer diameter fibers of polymer produced by electrospinning. Nanotechnology 1996;7:216-23.

Claims

1. A nanofiber comprising a water-soluble keratin polypeptide, at least one active ingredient and optionally at least one supporting polymer, said nanofiber being obtained by electrospinning an aqueous solution comprising said keratin polypeptide, said at least one active ingredient and optionally a supporting polymer.

2. The nanofiber according to claim 1 , wherein said aqueous solution further comprises a surfactant selected from anionic surfactants, cationic surfactants, non-ionic surfactants and mixtures thereof.

3. The nanofiber according to claim 1 or 2, wherein said supporting polymer is selected from the group consisting of polycaprolactone, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic acid), polyure thane, hyaluronic acid, polyhydroxy alkanoates (PHAs), polybutylene succinate (PBS), cellulose and derivatives thereof (methyl cellulose, hydroxymethylcellulose), starch, chitosan, fibroin, collagen, elastin, polyethylene glycol, polypropylene glycol, polymethyl methacrylate, prolamin, zein, pectin, casein, gluten, soy proteins, microalgae proteins, sericin, resilin, chitin, polyamides, polyethylene, polyethylene terephthalate, poly butylene terephthalate, polypropylene, polyvinylidene fluoride, polycarbonate, acrylonitrile butadiene styrene, polyvinyl chloride, polyvinyl fluoride and polyoxymethylene.

4. The nanofiber according to any one of claims 1 to 3, wherein said keratin polypeptide is selected from the group consisting of wild-type keratin polypeptides, chemically modified keratin polypeptides and mixtures thereof.

5. The nanofiber according to claim 4, wherein said keratin polypeptide has a molecular weight comprised between 150 kDa and 3.5 kDa, preferably 45 kDa.

6. The nanofiber according to any one of claims 1-5, wherein said nanofiber has a diameter comprised between 50 nm and 1000 nm, preferably comprised between 100 nm and 900 nm.

7. The nanofiber according to any one of claims 1-6, wherein said at least one active ingredient is in an amount comprised between 0.01% and 20% by weight of the total weight of the nanofiber.

8. The nanofiber according to any one of claims 1-7, comprising, in percentage by weight of the total weight of the nanofiber, from 0.1 to 99% of keratin polypeptide, from 0.01 to 20% of said at least one active ingredient and from 5 to 80% of said supporting polymer.

9. The nanofiber according to any one of claims 1-8, wherein said at least one active ingredient is selected from the group consisting of therapeutic agents, biologically active molecules, diagnostic agents, nutraceuticals, cosmetic ingredients, phytotherapeutic agents, natural products dyes, cosmeceuticals, vitamins and trace elements.

10. The nanofiber according to claim 9, wherein said at least one active ingredient is preferably a therapeutic agent, a biologically active molecule or a diagnostic agent selected from the group consisting of anti inflammatory agents, anticancer drugs, antimicrobial agents, analgesics, hormones, local and general anaesthetic agents, bone regeneration agents, osteoinductive agents, osteoconductive agents, biomaterials for osteogenesis, demineralized bone matrix, collagen, tricalcium phosphate, hydroxyapatite, coraline bone substitutes, bioactive glasses, glass ionomers, antianginals; anti-arrhythmic drugs; antibacterial and antiprotozoal agents; anti-coagulants; antidepressants; anti-diabetic drugs; anti-epileptic drugs; antifungal agents; antihistamines; anti-hypertensive drugs; anti-muscarinic agents; antineoplastic agents; anti-migraine drugs; anti-parasitic agents; antiparkinson drugs; antipsychotic, hypnotic and sedative agents; anti-stroke agents; anti-thrombotic agents; antitussives; antivirals; beta-adrenoceptor blocking agents; calcium channel blockers; cardiac inotropic agents; contraceptive agents; corticosteroids; dermatological agents; diuretics; gastro-intestinal agents; haemostatics; local anaesthetics; opioid analgesics; parasympathomimetics; peptides; steroids; stimulating agents; vasodilators, preferably anti-inflammatory drugs, drugs for dermatological diseases, antifungal for dermatological use, emollients and protectives for the treatment of wounds and ulcerations, antipruritics, including antihistamines and anesthetics, antipsoriatic agents, antibiotics and chemotherapeutics for dermatological use, corticosteroids, antiseptics and disinfectants, anti-acne agents.

1 1. A pharmaceutical composition comprising nanofibers according to any one of claims 1- 10, and a pharmaceutically acceptable carrier.

12. A pharmaceutical formulation for administration by enteral, particularly oral and rectal, route, parenteral, particularly subcutaneous and intradermal, route, transcutaneous route, sublingual and transmucosal, particularly ocular, nasal, vaginal and urethral, route, comprising the composition according to claim 1 1.

13. The pharmaceutical formulation according to claim 12 for use in the treatment of dermatological diseases, neoplastic diseases, neurodegenerative diseases, metabolic diseases, cardiovascular diseases and for bone reconstruction.

14. The pharmaceutical formulation according to claim 12 or 13, for administration by oral route, characterized in that it is in the form of a tablet or pill comprising a core and a coating, the coating consisting of nanofibers according to any one of claims 1-9.

15. A cosmetic formulation comprising nanofibers according to any one of claims 1-9, wherein said active ingredient is a cosmetic ingredient, a nutraceutical, a phytotherapeutic agent, a natural product, a dye, a cosmeceutical, a vitamin or a trace element, and a cosmetically acceptable vehicle.

16. The formulation according to any one of claims 12- 13, for transdermal or topical application, wherein said nanofibers form a membrane.

17. The formulation according to any one of claim 15, for transdermal or topical application, wherein said nanofibers form a membrane.

18. The cosmetic formulation according to claims 15 and 17 for the treatment of keratinous substrates, such as skin, nails and hair.

19. The cosmetic formulation according to any one of claims 15, 17 and 18 for treating skin aging signs and / or improving the appearance of keratinous substrates.

20. A method for producing a nanofiber according to any one of claims 1- 10, comprising the steps of: - dissolving in water said water-soluble keratin polypeptide, said at least one active ingredient and optionally said supporting polymer;

- electrospinning the thus-obtained solution.