阅读说明：本技术 包含丝心蛋白的纳米纤维以及包含水凝胶和所述纳米纤维的系统 (Nanofibers comprising fibroin and systems comprising hydrogel and said nanofibers ) 是由 维塔·瓜里诺 艾达·波滕扎 瓦莱里娅·里泽洛 于 2018-04-04 设计创作，主要内容包括：本发明涉及包括外膜(2)和芯(3)的纳米纤维(1),其中,所述外膜(2)由丝心蛋白制成,且所述芯(3)由生物相容性且可生物降解的聚合物制成。本发明还涉及获得所述纳米纤维的方法及其在输送生物活性分子和/或颗粒和/或细胞和/或治疗疾病中的用途。本发明还涉及所述粉状纳米纤维,可选择地分散在水溶液中,且涉及一种包含所述粉状纳米纤维的水凝胶和所述粉状纳米纤维和所述系统用于输送生物活性分子和/或颗粒和/或细胞和/或治疗疾病中的用途。(The invention relates to a nanofiber (1) comprising an outer membrane (2) and a core (3), wherein the outer membrane (2) is made of fibroin and the core (3) is made of a biocompatible and biodegradable polymer. The invention also relates to a method for obtaining said nanofibres and to the use thereof for delivering biologically active molecules and/or particles and/or cells and/or for treating diseases. The invention also relates to said powdered nanofibres, optionally dispersed in an aqueous solution, and to a hydrogel comprising said powdered nanofibres and to the use of said powdered nanofibres and of said system for the delivery of bioactive molecules and/or particles and/or cells and/or for the treatment of diseases.)

1. A nanofiber (1) comprising an outer membrane (2) and a core (3), characterized in that the outer membrane (2) is made of fibroin and the core (3) is made of a biocompatible and biodegradable polymer.

2. The nanofiber (1) according to claim 1, wherein the silk fibroin is a natural or recombinant silk protein, preferably selected from the group comprising silk fibroin obtained from domesticated worms (Bombyx mori), wild worms (Antheraea), species: Antheraea pernyi (Pernyi), Bombyx mori (yamamai), Indian tussah (Militta), amber (assama), etc.; Allium (Philosamia), species: Ricinus ricinus (Cynthia ricini)), Aranea (Araneae), Hymenoptera, Diptera, or obtained by DNA recombination techniques.

3. The nanofiber according to claim 1 or 2, wherein the biocompatible and biodegradable polymer is a natural or synthetic polymer, used alone or in combination.

4. A nanofiber as claimed in any one of claims 1 to 3 wherein the polymer is a water-soluble polymer.

5. A nanofiber according to any one of claims 1 to 4, wherein the polymer is selected from one or more of the group comprising polyethylene oxide (PEO), Polycaprolactone (PCL), hyaluronic acid, gelatin, collagen, chitosan, alginate and albumin.

6. A nanofiber according to any one of claims 1 to 5, wherein the polymer is PEO.

7. A nanofiber according to any one of claims 1 to 6, wherein the diameter of the nanofiber is 50 to 2000nm, or 200 to 600 nm.

8. The nanofiber according to any one of claims 1 to 7, wherein the nanofiber has a diameter of 200 to 500 nm.

9. The nanofiber according to any one of claims 1 to 8, wherein the outer film (6) has a thickness of 10 to 750nm, or 20 to 250 nm.

10. The nanofiber according to any one of claims 1 to 9, wherein the outer membrane (2) has a thickness of 200nm and the nanofiber has a diameter of 700nm, or the outer membrane (2) has a thickness of 100nm and the nanofiber has a diameter of 300nm, or the outer membrane (2) has a thickness of 100nm and the outer membrane has a diameter of 400nm, or the outer membrane (2) has a thickness of 60nm and the outer membrane has a diameter of 250 nm.

11. A nano-fiber according to any of claims 1 to 10, wherein the core (3) extends longitudinally, preferably over the entire length of the nano-fiber (1).

12. A nano-fiber according to any of claims 1 to 11, wherein the core (3) comprises one or more bioactive molecules and/or particles and/or cells.

13. The nanofiber according to claim 12, wherein the bioactive molecule is selected from the group comprising anti-tumor compounds, anti-coagulant compounds, anti-thrombotic compounds, antibodies, vaccines, antibiotics, anti-viral drugs, anti-inflammatory drugs, amino acids, peptides, proteins, enzymes, growth factors, angiogenic factors, nucleic acids (e.g. miRNA), salts, fibronectin, glycosaminoglycans, polysaccharides, vitamins, antioxidants, antimicrobials.

14. The nanofiber according to claim 12 or 13, wherein the bioactive molecule is selected from the group consisting of antineoplastic compounds (such as alkylating agents and analogues thereof, preferably cyclophosphamide, ifosfamide, chlorambucil, melphalan, estramustine, lomustine, carmustine, carboplatin, cisplatin, oxaliplatin, busulfan, troosufan, thiotepa, dacarbazine, procarbazine, temozolomide), antimetabolites (preferably methotrexate, raltitrexed, pemetrexed, fluorouracil, capecitabine, cytarabine, gemcitabine, tegafur, fludarabine, cladribine, mercaptopurine, thioguanine, pentostatin, clofarabine, nerabine), cytotoxic antibiotics (preferably daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, amsacrine, bleomycin, and analogues thereof), Actinomycin, mitomycin), derivatives of natural origin (preferably paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, irinotecan, topotecan, trabectedin, etoposide, trabectedin), hormones and antagonists (preferably diethylstilbestrol, ethisterone, medroxyprogesterone, megestrol, goserelin, leuprorelin, triptorelin, lanreotide, octreotide, tamoxifen, toremifene, fulvestrant, cyproterone, flutamide, bicalutamide, anastrozole, letrozole, exemestane), protein kinase inhibitors (preferably dasatinib, erlotinib, imatinib, nilotinib, sunitinib, sorafenib), monoclonal antibodies (preferably panitumumab, trastuzumab, rituximab, alemtuzumab, bevacizumab).

15. The nanofiber according to claim 12 or 13, wherein the bioactive molecule is selected from the group comprising hydrophilic small molecules.

16. The nanofiber according to claim 15, wherein the small molecule belongs to the group of antibiotics with cytotoxic action, such as mitomycin C, doxorubicin, epirubicin, gemcitabine.

17. The nanofibres according to claim 12, wherein the particles are preferably of inorganic, organic or hybrid organic-inorganic type.

18. The nanofiber according to claim 12, wherein the cell is a stem cell, a primary cell or a lineage cell in a native or engineered state.

19. A method for obtaining a nanofibre according to any of claims 1 to 18, characterised in that the method comprises the following steps, not necessarily in the order indicated:

a) providing a fibroin Solution (SF) with a concentration of 2% -14% w/w in formic acid;

b) providing an aqueous solution of a water-soluble polymer;

c) optionally, adding said biologically active molecules and/or particles and/or cells to said aqueous solution;

d) filling at least two syringes, at least one first syringe being filled with the fibroin solution and at least one second syringe being filled with the water-soluble polymer solution;

e) connecting the injector to a pumping system of a coaxial electrospinning system;

e) the electrospinning process was started.

20. The method according to claim 19, wherein the concentration of the fibroin Solution (SF) in formic acid is 2% to 14%, or 5% to 12%, or 6% to 12%, or 5% to 10%, or 7% to 9% w/w, the formic acid preferably being pure formic acid.

21. The method according to claim 19 or 20, wherein the aqueous solution is saline or a culture medium or a concentrated aqueous solution, or water, preferably redistilled water.

22. The method according to claim 19 or 20, characterized in that the electrospinning process is carried out by setting the parameters within the following ranges:

-a flow rate of 0.1 to 10mL/h, preferably 0.5 to 1 mL/h;

working distance between spinneret (cathode) and metal collector (anode): 5-100 cm, preferably 10-80 cm;

-potential difference between cathode and anode: 5-100 kV, preferably 15-60 kV.

23. A process according to claim 22, characterized in that the cathode voltage is-10 kV and the anode voltage is +10 kV; or the cathode voltage is-5 kV, and the anode voltage is +15 kVA; or the cathode voltage is-1 kV, and the anode voltage is +19 kV; or the cathode voltage is 0kV, and the anode voltage is +24 kV.

24. A method according to any of claims 19 to 23, wherein the electrospinning process is carried out by operating under a hood with suction activated using a metal collector coated with a removable layer to which the electrospun fibres do not adhere, such as an aluminium layer, a polymer film layer or a fabric.

25. The method of claim 19, comprising:

-providing a fibroin solution with a concentration of 6% to 12%, preferably 8% to 10% w/w in pure formic acid;

-providing a PEO solution in water, preferably in redistilled water, at a concentration of 2% to 6%, preferably 2.5% to 5% w/w;

-optionally, dissolving one or more water-soluble small molecules in the water;

-filling two syringes with the solution and connecting them to the pumping system of the coaxial electrospinning system;

-setting the parameters in the following ranges, starting the electrospinning process: flow rate: 1-0.5 mL/h; working distance between spinneret (cathode) and metal collector (anode): 17-19 cm, preferably 18 cm; working voltage: keeping the anode at 0kV, and keeping the cathode at 22-26 kV.

26. The method of any one of claims 19 to 25, further comprising a product stabilization treatment step.

27. The method according to claim 26, wherein the stabilizing treatment step is a chemical treatment comprising soaking the product obtained by electrospinning in an alcohol and/or water bath for at least 10 min.

28. The method according to claim 26 or 27, wherein in the stabilizing treatment step the alcohol is selected from methanol and ethanol, preferably ethanol, wherein the concentration of ethanol in the bath is 50-100% v/v.

29. The method according to claim 27 or 28, wherein in the stabilizing step, the soaking lasts 20-90 min, preferably 20-40 min, or 30 min.

30. The method of claim 27, wherein the product is soaked in methanol for about 30 min.

31. The nanofiber according to any one of claims 1 to 18, which is in a powdery form and has a particle size of 1 to 5000 μm, preferably 10 to 1000 μm, or 100 to 1000 μm.

32. An aqueous solution comprising the powdered nanofibers of claim 31.

33. A system which is a hydrogel comprising the powdered nanofibers of claim 31.

34. The system of claim 33, wherein the concentration of the powdered nanofibers is between 0.01% and 5% w/v, or between 0.01% and 1% w/v, or between 0.1% and 0.8% w/v, or between 0.2% and 0.5% w/v.

35. The system of claim 33 or 34, wherein the hydrogel comprises hyaluronic acid + chondroitin sulfate.

36. The system of any one of claims 33 to 35, wherein the hydrogel comprises one or more bioactive molecules and/or particles and/or cells.

37. The system of claim 36, wherein the one or more bioactive molecules are selected from the group consisting of anti-tumor compounds, anticoagulant compounds, anti-thrombotic compounds, antibodies, vaccines, antibiotics, anti-viral agents, anti-inflammatory agents, amino acids, peptides, proteins, enzymes, growth factors, angiogenic factors, nucleic acids (e.g., miRNA), salts, fibronectin, glycosaminoglycans, polysaccharides, vitamins, antioxidants, antimicrobials.

38. The system according to claim 36, wherein said nanofibers and/or said hydrogel comprise an active ingredient, preferably selected from the group comprising mitomycin C, doxorubicin, epirubicin, gemcitabine.

39. A method of obtaining the system of any one of claims 33 to 38, comprising:

-providing a nanofiber as claimed in any one of claims 1 to 18;

-grinding said nanofibres to obtain a powder having a particle size of 1 to 5000 μm, preferably 10 to 1000 μm, or 100 to 1000 μm;

-optionally, recrystallizing;

-dispersed in the hydrogel, optionally with the addition of one or more bioactive molecules and/or particles and/or cells.

40. Use of the nanofibers of any one of claims 1 to 18, 31 and 33 to 38 in the treatment of human or animal diseases.

41. Use of nanofibers according to claim 40, wherein said nanofibers and/or said hydrogel are used as carriers for biologically active molecules and/or particles and/or cells.

42. Use of nanofibres according to claim 40 or 41, characterised in that the use is tissue regeneration.

43. Use of nanofibers according to claim 40 or 41, for the treatment of hernias and prolapses, reconstruction of prostheses (e.g. mammary prostheses), as peripheral nervous system, vascular system (e.g. veins, arteries, arteriovenous fistulas for vascular access), lymphatic system (e.g. lymphatic circulation system, lymph nodes), cardiovascular system (e.g. coronary arteries and cardiac muscle), central nervous system (e.g. spinal cord, skin and its layers), tissue for housing and protecting internal organs (e.g. dura mater, pericardium, pleura peritoneum), tissue of the musculoskeletal system (e.g. tendons, ligaments, muscle, bone, cartilage, diaphragm), respiratory system (e.g. nasal mucosa, trachea, larynx, pharynx, bronchi, lung), digestive system (e.g. oesophagus, stomach, intestine, anal canal tissue), The tissue and mucosa of the oral cavity (e.g., gums, teeth, tongue), the tissues and organs of the urinary system (e.g., kidney, ureter, bladder, urethra, adrenal gland), the genitalia and the reproductive system (e.g., corpus cavernosum, prostate, uterus, vulva, vagina, endometrium, fallopian tube) are regenerated.

44. Use of nanofibers according to any one of claims 40 to 43, for the treatment of bladder cancer.

45. Use of nanofibers according to claim 40, wherein said nanofibers are in a hydrogel and said system comprises the active ingredient mitomycin C.

Background

Fibroin is a natural protein fiber produced by various insects (lepidoptera), spiders (arachnids), and various hymenoptera, diptera, coleoptera, and the like, and is generally produced by silkworms. Silk has attracted much attention as a drug carrier because of its biocompatibility, controllability of degradation process, and excellent ability to maintain the functionality of its loaded active ingredients.

Electrospinning is a common method by which nanofibers can be obtained from polymer solutions. This method is initially disclosed in US 1,975,504.

Alessandrino et al in Eng. Life Sci.2008,8, No.3,219-225 discloses the preparation of nanofibers from fibroin by electrospinning. Electrostatic spinning is performed on fibroin using solvents such as Hexafluoroisopropanol (HFIP), Hexafluoroacetone (HFA), and formic acid, which are toxic to humans and the environment (Zarkoob S et al, Structure and biology of electrospark silk fibers. Polymer 2004; 45: 3973-3977, KawaharaY et al, Structure for electro-sport-spill silk fibers. J Appl Polymer Sci 2008; 107: 3681-3684. Ohgo K et al.

WO2009042829 discloses nanofibers dispersed in a hydrogel, the nanofibers having a length on the order of nanometers or millimeters, consisting of cross-linked carboxyl-functional and hydroxyl-functional polymers, such as polyacrylic acid (PAA) and polysaccharides (e.g., dextran).

Nanofibers derived from fibroin are dispersed in hydrogels, for example, Elia R et al.j Biomater Appl 201327: 749. The polymer used is typically hyaluronic acid.

Synthetic polymers, such as polyethylene oxide (PEO), have been used in combination with chitosan to obtain nanofibers (Pakravan M et al, Biomacromolecules 2012,13, 412-.

There is a great need to develop a molecular carrier that is biocompatible, pharmacologically meaningful, and easily available and injectable.

Disclosure of Invention

The invention firstly provides a nanofiber (1) comprising an outer membrane (2) and a core (3), wherein the outer membrane (2) is made of fibroin and the core (3) is made of a biocompatible and biodegradable polymer, preferably the polymer is water-soluble.

In a preferred embodiment, the core of the nanofiber further comprises one or more bioactive molecules and/or particles and/or cells.

A second aspect of the invention is a method for obtaining said nanofibres, as shown in figure 8.

In another aspect, the powdered nanofibers (1) are disclosed and claimed, by powdered nanofiber is meant nanofibers having a particle size of 1 to 5000 μm, more preferably 10 to 1000 μm.

In another aspect, a hydrogel system comprising the nanofibers (1) is disclosed and claimed, wherein the nanofibers are present in the hydrogel in a powdered form. In a preferred embodiment, the system further comprises one or more bioactive molecules and/or particles and/or cells. The bioactive molecule and/or particle and/or cell is in the core of the nanofiber, or in the core of the nanofiber and in the hydrogel itself, or in the hydrogel.

The invention also relates to a method for obtaining said system.

Also claimed is the use of the nanofibers, or the powdered nanofibers, or the hydrogel system comprising the powdered nanofibers, optionally loaded with one or more bioactive molecules and/or particles and/or cells, and/or the hydrogel in the treatment of a disease. The nanofibres, or the powdered nanofibres or the system are administered and positioned in the area to be treated, allowing the controlled release of bioactive molecules and/or particles and/or cells consisting of biocompatible and biodegradable material, optionally contained within and re-taken.

Drawings

FIGS. 1 to 6: a graph of the nanofibers provided by the present invention obtained in example 1;

FIG. 7: schematic representation of nanofibers provided by the present invention;

FIG. 8: the invention provides a flow chart for obtaining nano fibers;

FIG. 9: the invention provides a flow chart for obtaining a hydrogel and nanofiber system.

Detailed Description

In a first aspect, with reference to fig. 7, a nanofiber (1) comprising an outer membrane (2) and a core (3) and a method for obtaining the nanofiber are described below, wherein the outer membrane (2) is made of fibroin and the core (3) is made of a biocompatible and biodegradable polymer.

The silk fibroin is a natural or recombinant silk protein. For example, it is obtained from domesticated worms (bombyx mori), wild insects (Antheraea, species: Antheraea pernyi, Bombyx mori (yamamai), Antheraea pernyi (militta), Acipenser succina (assama), etc.; Simarouba (Philosamia), species: Castor neriis sativus (cynthia ricini), etc., spiders (different species of Araneae), Hymenoptera insects (Hymenoptera), Diptera insects (dipterans), or by DNA recombination techniques known to those skilled in the art.

In a preferred embodiment, the polymer is selected from natural or synthetic biocompatible and biodegradable polymers, alone or in combination. Preferably, the polymer is water soluble. For example, it is selected from the group comprising polyethylene oxide (PEO), polylactic acid (PLA), polyglycolic acid (PGA), PLA-PGA combinations or PLGA copolymers, Polycaprolactone (PCL), hyaluronic acid, gelatin, collagen, chitosan, alginate, albumin. More preferably, the polymer is PEO.

Preferably, the diameter of the nanofiber is 50-2000 nm. In a preferred embodiment, the diameter of the nanofibers is 200 to 600nm, preferably 200 to 500nm, wherein the diameter corresponds to the sum of the thickness of the core (3) and the thickness of the outer membrane (2).

Preferably, the thickness of the outer film is 10 to 750nm, or 20 to 250 nm. For example, the outer membrane (2) of the nanofiber (1) provided by the invention has the thickness of 200nm and the total diameter of 700 nm. In addition, the thickness of the outer membrane (2) of the nanofiber is 100nm, and the total diameter is 300nm, or the thickness of the outer membrane (2) is 100nm, and the total diameter is 400nm, or the thickness of the outer membrane (2) is 60nm, and the total diameter is 250 nm.

The core (3) extends in the longitudinal direction, preferably over the entire length of the nanofibres (1) themselves.

In a preferred embodiment, the nanofibers comprise one or more bioactive molecules and/or particles and/or cells. The biologically active molecule is for example selected from the group comprising anti-tumour compounds, anti-coagulant compounds, anti-thrombotic compounds, antibodies, vaccines, antibiotics, anti-viral drugs, anti-inflammatory drugs, amino acids, peptides, proteins, enzymes, growth factors, angiogenic factors, nucleic acids (such as miRNA), salts, fibronectin, glycosaminoglycans, polysaccharides, vitamins, antioxidants, antimicrobial agents. For example, the antineoplastic agent may comprise an alkylating agent and analogs thereof (cyclophosphamide, ifosfamide, chlorambucil, melphalan, estramustine, lomustine, carmustine, carboplatin, cisplatin, oxaliplatin, busulfan, troosufen, thiotepa, dacarbazine, procarbazine, temozolomide), antimetabolites (methotrexate, raltitrexed, pemetrexed, fluorouracil, capecitabine, cytarabine, gemcitabine, tegafur, fludarabine, cladribine, mercaptopurine, thioguanine, pentostatin, clofarabine, nelarabine), cytotoxic antibiotics (daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, amsacrine, bleomycin, actinomycin, mitomycin), derivatives of natural origin (paclitaxel, docetaxel, vinblastine, vincristine, vinblastine, dactinomycin), Vindesine, vinorelbine, irinotecan, topotecan, trabectedin, etoposide, trabectedin), hormones and antagonists (diethylstilbestrol, ethinylestradiol, medroxyprogesterone, megestrol, norethindrone, goserelin, leuprorelin (leuproreline), triptorelin, lanreotide, octreotide, tamoxifen, toremifene, fulvestrant, cyproterone, flutamide, bicalutamide, anastrozole, letrozole, exemestane), protein kinase inhibitors (dasatinib, erlotinib, imatinib, nilotinib, sunitinib, sorafenib), monoclonal antibodies (panitumumab, trastuzumab, rituximab, alemtuzumab, bevacizumab).

In a preferred embodiment, the one or more biologically active molecules are selected from the group comprising water-soluble small molecules, more preferably belonging to the group of antibiotics with cytotoxic action. For example, mitomycin C, doxorubicin, epirubicin, gemcitabine are particularly suitable active ingredients for loading into the nanofibers according to the present invention.

The particles are preferably: submicron particles of inorganic type: quantum dots, magnetic nanoparticles, ceramics, and metals (e.g., platinum, palladium, rhodium, gold, silver, copper-based); organic type of submicron particles: liposomes, micelles, dendrimers, polymeric nanoparticles (PLA, PLGA, PVP, PEG, PCL, etc.) and nanogels (PAA, PVP, PVA, alginate, chitosan, collagen, fibrin, hyaluronic acid); inorganic-organic hybrid submicron particles. In a preferred embodiment, the particles are loaded with one or more of the aforementioned bioactive molecules and are contained in the polymer forming the core of the nanofibers of the present invention.

The cell is a stem cell, a primary cell, or a lineage cell in its native or engineered state. Preferably, the cell is a stem cell, stromal cell, embryonic cell, endothelial cell, epithelial cell, muscle cell, fibroblast, chondrocyte, osteoblast, leukocyte, lymphocyte or embryonic stem cell, such as an iPSC. For example, the cells are engineered with genes encoding growth factors.

In a preferred embodiment, the nanofibers are produced by an electrospinning process. The nanofibres are characterised in that they are obtained by coaxial electrospinning and by varying the concentration of the solution used and the process parameters, nanofibres with different diameters and with different ratios of core (3) thickness to outer membrane (2) thickness are obtained.

The coaxial electrospinning process comprises the following steps (not necessarily in the order indicated):

a) providing a fibroin Solution (SF) with a concentration of 2% -14% w/w in formic acid;

b) providing an aqueous solution of a water-soluble polymer;

c) optionally, adding said biologically active molecule and/or cell to said aqueous solution;

d) filling at least two syringes, at least one first syringe being filled with the fibroin solution and at least one second syringe being filled with the water-soluble polymer solution;

e) connecting the injector to a pumping system of a coaxial electrospinning system;

f) the electrospinning process was started.

In a preferred embodiment, the method comprises:

(a) providing a fibroin Solution (SF) with a concentration of 2% -14% or 5% -12% or 6% -12% or 5% -10% or 7% -9% w/w in formic acid, preferably pure formic acid. Said fibroin solutions having a high concentration are generally obtained from commercially available fibroin aqueous solutions, typically at a concentration of 5% w/w, by solidifying the aqueous solution to obtain a fibroin film, pouring the aqueous solution onto a suitable carrier, and air drying in a fume hood. In a further example, the fibroin water solution is prepared starting from natural silk fibers that do not contain sericin using methods known to those skilled in the art for producing fibroin films. Then dissolving the obtained fibroin membrane in formic acid, and completing the dissolution within 30-120 min, preferably 60-120 min under stirring;

b) providing an aqueous solution of a water-soluble polymer, i.e. a salt solution or a culture medium or a concentrated aqueous solution, or water, preferably redistilled water;

c) optionally, adding said biologically active molecules and/or particles and/or cells to said aqueous solution;

d) filling at least two syringes, at least one first syringe being filled with the fibroin solution and at least one second syringe being filled with the water-soluble polymer solution;

e) connecting the injector to a pumping system of a coaxial electrospinning system, the connection being made before or after filling without requirement;

f) the electrospinning process was started by setting the parameters within the following ranges:

-flow rate: 0.1-10 mL/h, preferably 0.5-1 mL/h;

working distance between spinneret (cathode) and metal collector (anode): 5-100 cm, preferably 10-80 cm;

-potential difference between cathode and anode: 5-100 kV, preferably 15-60 kV.

Particularly advantageous operating conditions have been demonstrated: a voltage of-10 kV is provided at the cathode, a voltage of +10kV is provided at the anode, and a potential difference of 20kV is formed between the cathode and the anode; or a voltage of-5 kV is provided at the cathode and a voltage of +15kV is provided at the anode; or a voltage of-1 kV is provided at the cathode and a voltage of +19kV is provided at the anode; or a voltage of 0kV at the cathode and a voltage of +24kV at the anode.

In a preferred embodiment, the water-soluble polymer is PEO and the PEO solution has a concentration of 2% to 6%, preferably 2.5% to 5% w/w, and the solvent is water, preferably redistilled water, for example obtained by adding PEO powder to water and stirring at room temperature, preferably overnight.

Preferably, the electrospinning process is carried out by operating under a hood with suction activated, using a metal collector coated with a removable layer on which the electrospun fibres do not adhere, such as an aluminium layer, a polymer film or a fabric, preferably a static metal collector coated with an aluminium layer.

In a more preferred embodiment, the electrospinning process is carried out with the following parameters:

-flow rate: 1-0.5 mL/h;

working distance between spinneret (cathode) and metal collector (anode): 17-19 cm, preferably 18 cm;

-applying a voltage: the anode is kept at 0kV, and the cathode is kept at 22-26 kV.

Preferably, the concentration of SF is 10% and the concentration of PEO is 5%; the operation was carried out at a flow rate of 0.5mL/h, a working distance of 18cm and a voltage of 24 kV.

In a preferred embodiment, use is made of

Prototype coaxial electrospinning apparatus, supplied by SKE Advanced therapeutics, in which the collector was coated with an aluminium layer.

Preferably, the electrospinning process is followed by a stabilizing chemical treatment. The stabilization treatment comprises soaking the product obtained from the process in an alcohol bath, preferably in a water and alcohol bath. For example, the alcohol is selected from methanol and ethanol, preferably ethanol, wherein the concentration of ethanol in the bath is 50-100% v/v. The soaking lasts at least 10min, or 20-90 min, preferably 20-40 min, or 30 min. Subsequently, the resulting stabilized product was pulled and removed from the aluminum sheet on which it was deposited, and dried at room temperature.

The chemical stabilization treatment enables the nanofibers provided by the present invention to maintain the nanofiber structure even if immersed in water due to the crystallization process.

In said optional step c) of loading with one or more active ingredients and/or particles and/or cells, said one or more active ingredients and/or particles and/or cells are added to said aqueous solution, forming a suspension or solution (depending on the type of active ingredient or the presence or absence of cells).

Experiments with 0.1% rhodamine b (rhodamine b) aqueous solutions, the sole purpose of which was to monitor the localization of the active ingredient during the preparation, showed that the resulting nanofiber cores had typical rhodamine staining.

In another aspect, the invention described herein provides nanofibers in a powdered form. The nano-fibers are ground to obtain powder with the particle size of 1-5000 microns, and preferably 10-1000 microns. In one embodiment, the grinding is done manually, in a mortar immersed in liquid nitrogen, to obtain particle sizes of 100-1000 μm with large differences between particle sizes, or by using a mortar grinder (mortar grinder) to obtain smaller particle sizes with small differences between particle sizes. Alternatively, methods known to those skilled in the art are used, such as mills (mills) (e.g., ball mills), or micronizers (e.g., round chamber air jets, elliptical chamber air jets, opposed air jet micronizers).

The powder thus obtained is collected and preferably recrystallized. The recrystallization treatment comprises soaking the product resulting from the process in an alcohol bath, preferably a water and alcohol bath. The alcohol is selected from, for example, methanol and ethanol, more preferably ethanol, wherein the concentration of ethanol in the bath is 50-100% v/v. The soaking lasts at least 10min, or 20-90 min, preferably 20-40 min, or 30 mm. The recrystallization is carried out, for example, by collecting the powder in methanol and drying the suspension, preferably at room temperature, in a vessel with a large surface. Subsequently, the dried product is repeatedly washed with water, preferably 2 or 3 times with redistilled water, stirred and centrifuged to eliminate any residual solvent. And then sterilized. The sterilization process may use ethylene oxide, gamma ray exposure, beta ray exposure, ultraviolet ray exposure; ultraviolet exposure is preferred.

When the nanofibers have been loaded with an active ingredient of a hydrophilic small molecule, it has been demonstrated herein that the active ingredient of the hydrophilic small molecule remains in the milled nanofibers as evidenced by the milling of the nanofibers comprising rhodamine B dye.

In another aspect, hydrogel systems comprising nanofibers provided herein and methods of making the systems are disclosed.

The hydrogel is selected from the hydrogels known in the background art. In embodiments where the system is developed for use in the bladder, the hydrogel is preferably composed of hyaluronic acid and chondroitin sulfate. This is because the urothelium is coated with a glycosaminoglycan and the coating (damaged, for example, by removal of the tumour) can be advantageously replaced by hyaluronic acid and chondroitin sulphate in the hydrogel, indicating that the latter is tropic for the glycosaminoglycan. In addition, hyaluronic acid is an adhesion agent for CD44 molecules expressed by tumor cells, and it can act as a chelating agent for residual tumor cells after tumor resection surgery in the bladder.

The method for obtaining a hydrogel + nanofibrous system according to the invention comprises obtaining powdered nanofibres, optionally recrystallised and sterilised as described before, and finally dispersing said powdered nanofibres in a solution of the selected hydrogel, preferably sterile.

In one embodiment, the nanofibers are dispersed in the hydrogel at a concentration of 0.01% to 5% W/v, or 0.01% to 1% W/v, or 0.1% to 0.8% W/v, or 0.2% to 0.5% W/v. Concentrations higher than the preferred concentration may make the system too firm to deliver in the form of a gel.

In embodiments where the hydrogel comprises one or more active molecules and/or particles and/or cells, they are dissolved/dispersed in the hydrogel in which the powder is dispersed in the desired amount.

In another embodiment, the powdered nanofibers are dispersed in an aqueous solution. The invention therefore also relates to an aqueous solution comprising said pulverulent nanofibres.

The invention also relates to the use of said nanofibers, and/or said powdered nanofibers dispersed in an aqueous solution, and/or said powdered nanofibers dispersed in a hydrogel for the delivery of active ingredients and/or for use as a carrier for tissue regeneration, wherein said nanofibers and/or said hydrogel are optionally loaded with one or more active molecules and/or particles and/or cells.

For example, there is evidence to support that the non-powdery nanofibers provided by the present invention can be advantageously used in the treatment of hernia and prolapse, in the reconstruction of prostheses such as mammary prostheses and as peripheral nervous system (nerves), vascular system (veins, arteries, arteriovenous fistulas for vascular access), lymphatic system (lymphatic circulation system, lymph nodes), cardiovascular system (coronary arteries and cardiac muscle), central nervous system (spinal cord), skin and its layers, tissues for housing and protecting internal organs (dura mater, pericardium, pleura, peritoneum, etc.) and musculoskeletal system tissues (tendons, ligaments, muscles, bones, cartilage, diaphragm), respiratory system (nasal mucosa, trachea, larynx, pharynx, bronchi, lung), digestive system (esophagus, stomach, intestine, anal canal tissues), oral tissue and mucosa (gums, teeth, etc.), tongue, urinary system (kidney, anal canal tissues), oral tissue, teeth, etc.) Ureters, bladder, urethra, adrenal gland), genitals, and tissues and organs of the reproductive system (corpus cavernosum, prostate, uterus, vulva, vagina, endometrium, fallopian tubes).

Also, the nanofibers, which are powdered and/or dispersed in an aqueous solution and/or incorporated into a hydrogel, may be included in a system and/or scaffold for repairing and regenerating all of the tissues listed in the preceding paragraph.

The nanofiber-based systems provided by the present invention (non-powdered, dispersed in aqueous solution, dispersed in hydrogel) can be used for regeneration following tissue and/or organ damage due to pathology, trauma or surgery, for example, in the case of total or partial resection with resection of a tumor.

In a preferred embodiment, the hydrogel is hyaluronic acid + chondroitin sulphate and the active ingredient is mitomycin C, the system being advantageously applied in the treatment of bladder cancer, in particular in the treatment after tumour resection.

In one embodiment, the present invention relates to a nanofiber (1) comprising an outer membrane (2) and a core (3), wherein the outer membrane (2) is prepared from fibroin and the core (3) is prepared from polyethylene oxide (PEO).

Preferably, the diameter of the nanofiber is 200-500 nm. Preferably, the core of the nanofiber comprises one or more hydrophilic molecules.

In an embodiment, the invention further relates to a method for obtaining nanofibers, wherein the method comprises the steps of:

-providing a fibroin Solution (SF) at a concentration of 6% to 12%, preferably 8% to 10% w/w in pure formic acid;

-providing a PEO solution having a concentration of 2% to 6%, preferably 2.5% to 5% w/w in water (preferably double distilled water);

-optionally, dissolving one or more water-soluble small molecules in said water;

-filling two syringes with the solution, connecting them to the pumping system of the coaxial electrospinning system;

-setting the parameters in the following ranges, starting the electrospinning process: flow rate: 1-0.5 mL/h, working distance between spinneret (cathode) and metal collector (anode): 17-19 cm, preferably 18cm, voltage: keeping the anode at 0kV and the cathode at 22-26 kV.

Preferably, the process further comprises a stabilization treatment step, in particular by immersing the product obtained from the previous process in methanol for about 30'.

In another embodiment, the invention relates to a hydrogel system comprising the nanofiber provided by the invention, and is characterized in that the nanofiber is powdery and has a particle size of 10-1000 μm. Preferably, the hydrogel comprises hyaluronic acid + chondroitin sulfate. Preferably, the nanofibers and/or the hydrogel comprise an active ingredient selected from the group comprising: mitomycin C, doxorubicin, epirubicin, gemcitabine.

In another embodiment, the invention relates to a method of obtaining the aforementioned system, comprising the steps of:

-providing a nanofiber as provided by the present invention;

-grinding said nanofibres to obtain a powder with a particle size of 10-1000 μm, preferably 100-1000 μm;

-optionally, recrystallization in methanol;

-dispersed in the hydrogel, optionally with the addition of one or more hydrophilic small molecules.

In another embodiment, the invention relates to the use of said system as a carrier for active ingredients and/or a scaffold for tissue regeneration. Preferably, the system comprises mitomycin C as an active ingredient for the treatment of bladder cancer.

The advantage of using fibroin over other biopolymers (e.g., chitosan) is apparent because fibroin, while biodegradable, is more resistant to aqueous environments.

In addition, fibroin has surprising biomimetic potential. This allows the nanofiber system provided by the present invention to act not only as a carrier for active ingredients, but also as a scaffold for tissue regeneration in itself, since the nanofibers provided by the present invention can be integrated into the system in which they are located.

Another advantage is that the mechanical resistance to grinding of fibroin is unexpectedly greater compared to other biopolymers. For example, chitosan-PEO nanofibers in the background art cannot be powdered according to the present invention because the powdering process destroys the nanofiber structure of chitosan-PEO nanofibers known in the background art. Only with the nanofibers provided by the present invention, powders that retain the nanofiber structure can be obtained.

Furthermore, fibroin has a programmable biodegradability, i.e. by varying the thickness of the outer membrane of the nanofibrils, the biodegradability of fibroin can be adjusted, thus controlling the release of the active ingredient dissolved in the core of the nanofibrils themselves. These features enable surprisingly controlled in vivo release kinetics, which allow a long time fine-tuning of the biodegradation of the fibroin and thus of the release of the active ingredient delivered by the system provided by the present invention.

It has been observed that the greater dimensional variability obtained by manually grinding the nanofibres favours the release of the active ingredient contained inside the nanofibres over time by a continuous system, since smaller fragments release more rapidly and larger fragments release with delay. Thus, a larger size distribution of the fragments may result in a better distribution of the release of the active ingredient over time.