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WO2025137557A1 - Membrane de nanofibres électrofilées pour transplantation de cellules cornéennes - Google Patents

Membrane de nanofibres électrofilées pour transplantation de cellules cornéennes Download PDF

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Publication number
WO2025137557A1
WO2025137557A1 PCT/US2024/061438 US2024061438W WO2025137557A1 WO 2025137557 A1 WO2025137557 A1 WO 2025137557A1 US 2024061438 W US2024061438 W US 2024061438W WO 2025137557 A1 WO2025137557 A1 WO 2025137557A1
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WO
WIPO (PCT)
Prior art keywords
membrane
cells
gelnf
corneal
cell
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Pending
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PCT/US2024/061438
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English (en)
Inventor
David Myung
Euisun SONG
Won-Gun Koh
Karen M. CHEN
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Industry Academic Cooperation Foundation of Yonsei University
US Department of Veterans Affairs
Leland Stanford Junior University
Original Assignee
Industry Academic Cooperation Foundation of Yonsei University
US Department of Veterans Affairs
Leland Stanford Junior University
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Publication of WO2025137557A1 publication Critical patent/WO2025137557A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/14—Eye parts, e.g. lenses or corneal implants; Artificial eyes
    • A61F2/142—Cornea, e.g. artificial corneae, keratoprostheses or corneal implants for repair of defective corneal tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3808—Endothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea

Definitions

  • the corneal endothelium the innermost layer of the cornea, is comprised of a single layer of corneal endothelial cells (CECs) maintaining a high cell density. It is anchored to Descemet’s Membrane (DM) which is approximately 10 ⁇ m in thickness.
  • CECs corneal endothelial cells
  • DM Descemet’s Membrane
  • Disruptions in this function may result in edema of the corneal stoma and surface epithelium.
  • Current methods for transplanting CECs include Descemet’s stripping automated endothelial keratoplasty (DSAEK) and descemet’s membrane endothelial keratoplasty (DMEK) as current clinical gold standards, while approaches undergoing clinical evaluation include direct injection and transplantation as sheets on fabricated carriers, mimicking DMEK transplant surgery. While direct cell injection approaches have shown promising results in clinical trials, they are currently not yet FDA-cleared and widely available for use in patients.
  • Electrospinning is a valuable tools for creating thin membranes with high permeability. It is capable of producing various forms of nanofiber scaffolds, and has been actively utilized in the field of tissue engineering, particularly in wound healing, vascular graft, and cardiac tissue engineering.
  • DMEK-like corneal transplantation procedure a 7 to 8 mm circular disc of Descemet’s membrane and attached endothelial cells are rolled into a cylinder before they are injected into the anterior chamber through a 2.4 mm-4.0 mm incision. The transplant graft is then unfurled and properly placed endothelial side down before adhering to the posterior cornea with the aid of a gas bubble.
  • DMEK is currently employed successfully by corneal surgeons with very good visual outcomes, but is limited by a higher learning curve and higher rates of post-operative re-bubbling to fully attach the graft compared to DSAEK.
  • Electrospun nanofiber membranes are provided.
  • the membranes are electrospun from a suitable biocompatible and biodegradable biopolymer, e.g. gelatin, collagen, etc., to generate a membrane that is then cross-linked.
  • the membrane thus generated has adjustable mechanical properties, and the thickness of nanofiber membrane. It features high permeability of biological factors and transparency in visible light wavelength.
  • the membrane compositions find use tissue regeneration, particularly in repair, regeneration, and/or reconstruction of lamellar or partial defects of wounded corneal tissue.
  • the nanofibers are formed of proteins such as collagen, or derivatives of collagen such as gelatin.
  • the nanofibers are cross-linked, e.g. with glutaraldehyde.
  • the membrane is a composite of collagen or gelatin protein, with additional biomolecule(s), e.g. glycosaminoglycans such as hyaluronic acid, chondroitin sulfate, heparan sulfate, and dermatan sulfate.
  • the protein and glycosaminoglycans can be physically or chemically crosslinked to each other.
  • a feature of the membranes of the disclosure is that it provides a scaffold with a short waiting time for cell attachment, e.g. from about 5 to about 15 minutes.
  • the nanofiber membrane is seeded with cells, e.g. corneal endotheial cells, etc.
  • the nanofiber membrane provides a delivery vehicle for corneal endothelial cell transplantation, which may be implanted after removal of diseased or damaged Descemet’s membrane and endothelial cells.
  • the nanofiber membrane provides a transplantable scaffold for corneal regeneration.
  • the nanofiber membrane provides a transplantable scaffold for corneal regeneration through the delivery of corneal cells.
  • the nanofiber membrane provides a carrier for ocular drug delivery.
  • the nanofiber membranes may have a thickness ranging from about 5 ⁇ m, about 10 ⁇ m, about 20 ⁇ m to about 150 ⁇ m.
  • Transparency is at least about 75% compared to glass, at least about 80% compared to glass, and may be at least about 85% compared to glass.
  • the calculated fiber diameter may be from about 200 to about 400 nm, or from about 250 to about 300 nm.
  • the elastic modulus may be similar to that of the human Descemet’s membrane, e.g. from about 0.2 to about 0.6 Mpa, and may be around about 0.3 to about 0.5 Mpa.
  • an electrospun membrane composition for use in treating or reconstructing a surgically incised or wounded corneal area in a mammalian subject in need thereof.
  • the electrospun membrane may comprise one or both of cells and therapeutic agents that aid in treating or reconstructing a surgically incised or wounded area, where the cells or agent are entrapped or encapsulated in the defined hydrogel structure.
  • Cells of interest include regenerative cells, such as a stem cell, including without limitation corneal stem cells, corneal stromal stem cells, corneal mesenchymal stromal cells, corneal limbal epithelial cells, corneal epithelial cells, corneal endothelial cells, keratinocytes, etc.
  • Cells suitable for treating corneal tissue may include, for example, one or more of corneal stromal stem cells, mesenchymal cells, keratocytes, keratinocytes, endothelial cells, and epithelial cells, and limbal epithelial cells, and transient amplifying cells.
  • a method of treating or reconstructing a surgically incised or wounded corneal site in a mammalian subject is provided, by administering a electrospun membrane to deliver cells, drugs, factors, etc.
  • a cavity is debrided to eliminate scarred, fibrotic, and/or necrotic material and create fresh wound edges.
  • a biocompatible hydrogel electrospun membrane is provided that optionally comprises cells, therapeutic agents, etc.
  • the biocompatible hydrogel structure is suitable for use in tissue repair or regeneration.
  • the membranes in the present invention can serve as, but are not be limited to, tissue scaffolds, tissue substitutes, optical elements (e.g. corneal or lens tissue), tissue fillers, tissue spacers, or as delivery vehicles for cells, tissues, and/or pharmaceutical agents.
  • FIGS.1A-1B Optimization of spinning time and crosslinking time based on thickness and transparency of the gelNF membrane.
  • A Macroscopic image of gelNF membrane under varied spinning and crosslinking times. The numbers on the top right of the image represent spinning time on the left of the slash and crosslinking time on the right of the slash.
  • FIGS. 2A-2C Analysis in nanofiber diameter and mechanical property of gelNF membrane.
  • A SEM images of the gelNF membrane in before/after GA crosslinking and
  • B calculation of the nanofiber diameter.
  • C Tensile stress test of gelNF membrane and calculated elastic modulus, elongation at break, and ultimate tensile strength.
  • FIGS.3A-3C Permeability and degradability of the gelNF membrane.
  • A Permeability test of the gelNF membrane using FITC-dextran and cell insert.
  • FIGS.4A-4C IhCEC culture on top of the gelNF membrane.
  • A Cytotoxicity of the gelNF membrane relative to the TCP and
  • B comparison of the morphology and cell density of the IhCEC cultured on TCP and the gelNF membrane on day 3 and 7.
  • Scale bar 200 ⁇ m.
  • FIGS.5A-5B PrCEC culture on top of the gelNF membrane.
  • A Morphology and Live & Dead staining of the PrCEC cultured on top of the gelNF membrane on day 1. Scale bar in phase contrast image: 100 ⁇ m and for the Live & Dead staining: 50 ⁇ m.
  • FIGS.6A-6F Ex vivo study for transplantation of PrCEC cultured on top of the gelNF membrane using geuder and artificial anterior chamber.
  • A Macro images for gelNF membrane placed inside of the geuder and schematic images for PrCEC cultured on top of the gelNF membrane transplantation.
  • FIGS.7A-7C Cytotoxicity of gelNF. Live & Dead assay of the IHECE cultured on top of the gelNF membrane on day (A) 3 and (B) 7. Scale bars : 50 ⁇ m.
  • C Quantified ratio of the live cell; [0026] FIGS.8A-8D.
  • FIGS.9A-9B Functional protein expression of PrCEC cultured on top of the gelNF membrane.
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • reconstructing and “reconstruction,” and the like are used herein to generally refer to rebuilding, healing and regenerating an injured matter or tissue.
  • subject or “mammalian subject” refers to any mammalian subject for whom treatment or therapy is desired, particularly humans.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as non-human primates, dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is a human.
  • the term "therapeutically effective amount” or “effective amount” means the amount of a compound, agent, composition, construct that when administered to a mammalian subject for treatment is sufficient, in combination with another agent, or alone in one or more doses or administrations, to effect such treatment for the disease.
  • a “biocompatible” substance for example polymer or cross-linking agent
  • a “biocompatible” substance is one that does not generally cause significant adverse reactions (e.g. toxic or antigenic responses) to cells, tissues, organs or the organism as a whole, of example, whether it is in contact with the cells, tissues, organs or the organism as a whole, for example, whether it is in contact with the cells, tissues, organs or localized within the organism, whether it degrades within the organism, remains for extended periods of time, or is excreted whole.
  • a biocompatible substance may be selectively compatible in that it exhibits biocompatibility with certain cells, tissues, organs or even certain organisms.
  • the biocompatible substance may be selectively biocompatible with vertebrate cells, tissues and organs but toxic to cells from pathogens or pathogenic organisms. In some circumstances, the biocompatible substance may also be toxic to cells derived from tumors and/or cancers.
  • nanofiber refers to a fiber with a diameter no more than 1000 nanometers, and may be less than about 500 nm, less than about 350 nm, less than about 300 nm, and may be from about 200 to about 35 nm in diameter.
  • biopolymer refers to a biocompatible polymers comprising polymers that can be found naturally in organisms, as well as chemical and physical modifications of such polymers, and include, but are not limited to, proteins, fibrins, fibrinogen, collagens, gelatins, elastins, laminin, fibronectin, extracellular matrix constituents, glycosaminoglycans, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, hyaluronic acid, albumin, alginates, chitosans, cellulose, thrombin, heparin,polysaccharides, synthetic polyamino acids, prolamines, combinations thereof, and other such molecules.
  • the biopolymer is a conjugated version of the native biopolymer, where the conjugate confers additional functionality such as crosslinkability.
  • conjugations include functionalization with, for instance, a photocrosslinkable moiety such as a methacrylate, acrylate, or methacrylamide, or a functional group involved in non-photochemical crosslinking reactions.
  • Other functional groups that can be conjugated to the biopolymers include but are not limited to thiols, norboronenes, vinyl sulfones, azide, alkynes, and the like.
  • polymer refers to a molecule consisting of individual monomers joined together. Polymers that are contemplated herein can be naturally occurring, synthetically produced, or produced using recombinant methodologies.
  • transparent refers to at least 70%, 80, or 90% transmission of white light.
  • Electrospinning is a versatile method of fabricating polymeric fibers. The polymer fibers are typically characterized by fiber diameters ranging from several microns down to 100 nm or less. These polymeric fibers may be used to further fabricate products of varying complexity and different three-dimensional shapes. Electrospun (ES) fibers can be cross-linked, e.g.
  • the term “electrospinning” refers to a process in which a high voltage is used to create an electrically charged jet of polymer fluid, such as a polymer solution, which dries or solidifies to generate polymer fibers.
  • Systems for electrospinning generally include a syringe, a nozzle, a pump, a high-voltage power supply, and a grounded collector.
  • a high voltage power supply is connected to the orifice of the needle at one end and to the grounded collector on the other end.
  • the methods generally include the use of an external electric field for atomization of the polymeric solution during the spinning process.
  • the solution source e.g., a needle used for injection of the solution into the spinning apparatus.
  • the surface tension of the droplet is in equilibrium with the electric field. Electrostatic atomization occurs when the electrostatic field is strong enough to overcome the surface tension of the liquid.
  • any suitable electric field can be used in the methods of the invention. Typically, electric fields ranging from around 100 V to around 100 kV are used in the methods of the invention. The electric field can range, for example, from 500 V to 50 kV, or from 1 kV to 25 kV, or from 5 kV to 15 kV.
  • the electric field can be 5 kV, 5.5 kV, 6 kV, 6.5 kV, 7 kV, 7.5 kV, 8 kV, 8.5 kV, 9 kV, 9.5 kV, 10 kV, 10.5 kV, 11 kV, 11.5 kV, 12 kV, 12.5 kV, 13 kV, 13.5 kV, 14 kV, or 15 kV.
  • Other field strengths can be used depending on the composition of the particular solution used for the electrospinning process.
  • Any suitable flow rate can be used for introducing the solution from the source into the electric field. Typically, the flow rate will range from about 0.1 mL/hr to about 5 mL/hr.
  • the flow rate can range, for example from 0.1 mL/hr to 0.5 mL/hr, or from 0.5 mL/hr to 1 mL/hr, or from 1 mL/hr to 1.5 mL/hr.
  • the flow rate can range from 0.5 mL/hr to 1.5 mL/hr, or from 0.5 mL/hr to 1 mL/hr.
  • the flow rate can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 mL/hr.
  • Other flow rates can be used depending on factors including the composition of the solution and the strength of the electric field used in the process.
  • the target surface used for fiber collection can be placed at any suitable position with respect to the source of the solution.
  • the distance between the solution source and the target surface will typically range from about 5 cm to 50 cm.
  • the distance can range, for example, from 5 to 10 cm, or from 10 cm to 15 cm, or from 15 cm to 20 cm, or from 20 cm to 25 cm.
  • the distance can range from 5 cm to 30 cm, or from 10 cm to 20 cm.
  • the distance between the solution source and the target surface can be around 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 cm. Other distances can be employed depending on factors including the composition of the solution, the strength of the electric field, and the solution flow rate used in the process.
  • the polymer product comprises a fiber mat, or membrane, which can provide a substrate for cell and/or tissue culture.
  • the term “cell” in the context of the in-vivo and in-vitro applications of the present invention encompasses mammalian cells of any genus or species, particularly human cells.
  • the types of cells that may be incorporated into the polymeric biomaterial include progenitor cells of the same type as those from the tissue site, and progenitor cells that are histologically different from those of the tissue site such as embryogenic or adult stem cells, that can act to accelerate the healing, regenerative or reconstructive process.
  • compositions comprising cells can be administered in the form of a solution or a suspension of the cells mixed with the polymeric biomaterial solution, such that the cells are substantially immobilized within the application site upon gelation. This serves to concentrate the effect of the cells at the site of application; and may provide for release of the cells over a course of time
  • Corneal cells may used, for example limbal epithelial stem cells, corneal stromal stem cells, keratinocytes, keratocytes, corneal endothelial cells, etc.
  • Corneal stem cells may be isolated, harvested, and/or propagated from cadaveric donor corneal tissue, from small limbal biopsies from patients (either autologous from a patient’s healthy eye, or from another living patient's eye as a donation); generated by in vitro culture, etc.
  • Corneal stem cells also include corneal stromal stem cells, which are quiescent, mesenchymal cells. It has been suggested that corneal stromal stem cells are a subpopulation of stromal cells that can differentiate into keratocytes. Stromal corneal stem cells are also positive for ABcG2 expression.
  • Corneal endothelial cells (CECs) play a pivotal role in maintaining the clarity and function of the cornea by regulating fluid balance and preventing edema.
  • human corneal endothelial cells are a monolayer of hexagonal- shaped cells located at the posterior surface of the cornea. Despite their low proliferative capacity in vivo, these cells exhibit remarkable functional efficiency in maintaining corneal transparency. Morphologically, human corneal endothelial cells form a tightly packed hexagonal mosaic, ensuring optimal coverage of the corneal surface.
  • crucial markers associated with CECs is Na+/K+-ATPase, an enzyme responsible for actively pumping ions across the cell membrane.
  • ZO-1 a tight junction protein that plays a pivotal role in cell-cell adhesion and the formation of the endothelial cell monolayer. ZO-1 not only contributes to the structural integrity of the endothelium but also participates in the regulation of paracellular permeability, ensuring the maintenance of corneal transparency. The success of corneal transplantation critically relies on the preservation, isolation, and transplantation of viable human corneal endothelial cells.
  • N-cadherin is a cell adhesion molecule that contributes to the hexagonal mosaic pattern characteristic of corneal endothelial cells.
  • Therapeutically effective amounts of the cells seeded on a membrane of the instant disclosure will vary depending e.g., on the condition to be treated, typical survival of the particular cell type within the hydrogel construct (e.g., including the average lifespan of cells of the particular cell type), etc.
  • a therapeutically effective amount of cells on a membrane of the disclosure is 1x10 3 or more cells/cm 2 , including e.g., 5 x l0 3 cells/cm 2 or more, 1 x l0 4 cells/cm 2 or more, 5 x l0 4 cells/cm 2 or more, 1 x l0 5 cells/cm 2 or more, 5 x l0 5 cells/cm 2 or more, 1 x l0 6 cells/cm 2 or more, 5 x l0 6 cells/cm 2 or more, 1 x l0 7 cells/cm 2 or more, 5 x l0 7 cells/cm 2 or more.
  • the term “under physiological conditions” encompasses those conditions that are compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, osmolarity, osmolality etc.
  • the nanofiber membranes may comprise suitable therapeutic factors.
  • Suitable growth factors and cytokines include, but are not limited to stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage stimulating factor (GM-CSF), stromal cell-derived factor- 1, steel factor, vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFP), platelet derived growth factor (PDGF), angiopoeitins (Ang), epidermal growth factor (EGF), fibroblast growth factor (FGF) hepatocyte growth factor, nerve growth factor, keratinocyte growth factor, insulin-like growth factor (IGF-1), interleukin (IL)-3, IL-la, IL- ⁇ , IL-6, IL-7, IL-8, IL-11, and IL-13, colony- stimulating factors, thrombopoietin, erythropoietin, fit3-ligand, and tumor necrosis factor ⁇ .
  • SCF stem cell factor
  • growth factors examples include EGF, bFGF, HNF, NGF, PDGF, IGF-1 and TGF. These growth factors can be mixed with the membrane materials comprising the compositions.
  • the bioactive agents can also have pro- angiogenic activities, e.g., VEGF, PDGF, prominin-1 polypeptide, and variants thereof that have pro-angiogenic activities, i.e., promote neovascularization and angiogenesis.
  • one or more factors is provided at a concentration ranging from about 0.01 mg/mL to about 10 mg/ml in the membrane hydrogel, including any concentration in this range such as about 0.01 mg/ml, 0.1 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml, 3 mg/ml, 3.5 mg/ml, 4 mg/ml, 4.5 mg/ml, 5 mg/ml, 5.5 mg/ml, 6 mg/ml, 6.5 mg/ml, 7 mg/ml, 7.5 mg/ml, 8 mg/ml, 8.5 mg/ml, 9.0 mg/ml, 9.5 mg/ml, or 10 mg/ml.
  • the factor(s) is at a concentration of at least 1 mg/mL in the membrane.
  • the factors from can be crosslinked to the biopolymer in the membrane using SPAAC.
  • factors can be conjugated for SPAAC with an azide-N-hydroxysuccinimide (NHS) crosslinker to produce an azide-conjugated secreted factor.
  • the biopolymer can be conjugated with an alkyne-NHS crosslinker to produce an alkyne conjugated protein, which is subsequently reacted with the azide conjugated factor, thereby crosslinking the biopolymer and the factor within the membrane.
  • a factor can be reacted with an alkyne-NHS crosslinker to produce an alkyne-conjugated factor.
  • a biopolymer can be reacted with an azide-NHS crosslinker to produce an azide-conjugated biopolymer, which is subsequently reacted with the alkyne-conjugated factor thereby crosslinking the biopolymer and the factor within the hydrogel.
  • Photochemical or non-photochemical bioconjugation methods can also be used for direct covalent linkage of factors to biopolymers. Factors may include more than one functional group that can be crosslinked to allow formation of bonds among factors and the membrane.
  • the nanofiber membranes may comprise drugs for deliver to the eye.
  • Suitable drugs include, without limitation, Glaucoma Medications such as Latanoprost (Xalatan), Timolol (Timoptic), Dorzolamide (Trusopt), Brimonidine (Alphagan), Travoprost (Travatan), Bimatoprost (Lumigan); anti-inflammatory agents such as Prednisolone (Pred Forte, Pred Mild), Dexamethasone (Maxidex), Fluorometholone (FML), Loteprednol (Lotemax); antibiotics for ocular infections such as Tobramycin (Tobrex), Ciprofloxacin (Ciloxan), Ofloxacin (Ocuflox), Besifloxacin (Besivance), Moxifloxacin (Vigamox); allergy medications such as Olopatadine (Patanol, Pataday), Ketotifen (Zaditor, Alaway), Azelastine (Optivar), Cyclosporine (Restasis,
  • the electrospun biomaterial compositions can be seeded with cells, i.e. cellularized. Such cells can be somatic/differentiated cells, pluripotent stem cells, or progenitor/stem cells.
  • the electrospun membrane compositions and methods of the present invention can be applied to any clinical situation where tissue engineering, regeneration or reconstruction in a mammalian host or subject is necessary. Tissue engineering is a rapidly growing field encompassing a number of technologies aimed at replacing or restoring tissue and organ function. The key objective in tissue engineering is the regeneration of a defective tissue through the use of materials that can integrate into the existing tissue so as to restore normal tissue function.
  • compositions can comprise cells that settle in the host and encourage recellularization of the wounded tissue. Furthermore, such compositions can also serve as a three-dimensional tissue model for the in-vitro study of cellular responses and interplay.
  • Application to corneal transplant [0066]
  • the compositions of the disclosure address a need for effective compositions and methodologies to treat cornal transplantation.
  • Ocular and corneal defects may be caused by, e.g., neurotrophic keratopathy, recurrent corneal erosion, corneal ulcer, corneal burns, exposure keratopathy, physical trauma, retinal disease, retinal degeneration, optic nerve damage, optic nerve degeneration, and other disorders.
  • Corneal endothelial cell disorders include Fuchs’ Endothelial Dystrophy and Pseudophakic Bullous Keratopathy.
  • Delivering (cultured) cells such as corneal limbal epithelial cells, corneal stromal stem cells (CSSCs) or corneal endothelial cells to the site of corneal injury may minimize the fibrotic response and enhance the regeneration of the corneal tissue.
  • Delivery of corneal cells, such as keratocytes and keratinocytes, and other cells, seeded on nanofiber membranes, as described herein, may be instrumental in repairing and regenerating corneal tissue.
  • kits comprising separate containers holding compositions comprising nanofiber membranes, and optionally seeded with living cells to be delivered to the wounded tissue site.
  • Compositions can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic.
  • the kit can further comprise a container comprising pharmaceutically acceptable excipients or formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices.
  • the kit can also comprise a package insert containing written instructions describing methods for care of a corneal wound as described herein.
  • the electrospun membrane compositions of the present invention can be administered in the form of pharmaceutical compositions, comprising an isotonic excipient prepared under sufficiently sterile conditions for administration to a mammalian subject, particularly to a human being.
  • pharmaceutical compositions comprising an isotonic excipient prepared under sufficiently sterile conditions for administration to a mammalian subject, particularly to a human being.
  • the corneal endothelium comprised of densely packed corneal endothelial cells (CECs) adhering to Descemet's membrane (DM), plays a critical role in maintaining corneal transparency by regulating water and ion movement.
  • CECs have limited regenerative capacity within the body and globally there is an insufficiency of donated corneas to replace damaged corneal endothelium.
  • DM Descemet's membrane
  • the fabricated gelNF membrane exhibited approximately 80% transparency compared to glass and maintained a thickness of 20 ⁇ m. Moreover, the gelNF membrane demonstrated desirable permeability and degradability. Importantly, CECs cultured on the gelNF membrane at high densities showed no cytotoxic effects, and the expression of cellular functional proteins was verified. To assess the potential of this gelNF membrane as a carrier for cultured CEC transplantation, we conducted Descemet’s membrane endothelial keratoplasty (DMEK) on rabbit eyes. The outcomes demonstrate this gelNF membrane is suitable as a carrier for cultured CEC transplantation, offering advantages in terms of transparency, permeability, and sufficient mechanical properties required for successful transplantation.
  • DMEK membrane endothelial keratoplasty
  • the gelatin solution (25%, w/v) was then placed into a 5 ml syringe and connected to a 23-gauge needle.
  • the Electrospinning machine (TL-01, Tong Li Tech) fabricated the nanofiber membrane using a flow rate of 1 ml/h, 17 kV of voltage, and a 15 cm distance from needle to collector.
  • the nanofibers were collected at the cover glass, covered with aluminum foil, and evaporated overnight at RT to remove residual solvent.
  • the samples were then crosslinked with glutaraldehyde (G6257, Sigma Aldrich) under a vapor crosslink system and evaporated overnight at RT to remove residual solvent.
  • the nanofiber membranes were soaked in PBS, then separated from the foil.
  • the fiber diameter was calculated from the 100 fibers of each group using image J.
  • 12.5 ug/ml of the dextran dissolved in the cell culture media were added at the top of the cell crown. After 24h incubation, the media from the bottom part of the cell crown were measured to calculate the penetrated dextran.
  • the gelNF membranes were incubated at 37°C incubation in PBS for 14 and 28 days and SEM employed to evaluate changes in the nanofiber.
  • the specimens were prepared in rectangular shape (5 cm x 5 cm).
  • the 30 mm/min strain was assessed with 1 kN load cell until break point.
  • the elastic modulus was calculated from the stress-strain curve.
  • IhCEC Immortalized human corneal endothelial cell (IhCEC) culture. IhCEC (T0577) purchased from Applied Biological Materials (ABM) and cultivated as followed by manufacture’s protocol.
  • the culture media was supplements with 10% FBS (Gibco), 5 ug/ml human insulin (TM058), 10 ⁇ g/ml human transferrin, 3 ng/ml sodium selenite, 10 nM hydrocortisone, 10 nM ⁇ -estradiol, 10 ng/ml rhVEGF 165aa (Z100895), 10 ng/ml rhEGF (Z100135), 10 ng/ml Heparin, 2 mM L-glutamine (G275), and 1% Penicillin/Streptomycin Solution (15140122, Gibco).
  • the IhCECs were cultivated on substrate coated with FNC coating mix (Athena (0407, Athena)) 37°C in a humidified incubator with 5% CO2 and subcultured using 0.05% Trypsin-EDTA (Gibco). The culture medium was changed every 2-3 days.
  • Isolated primary rabbit corneal endothelial cell (PrCEC) culture Rabbit eyeballs were purchased from Visiontech inc.
  • descemet’s membrane was separated and placed in 2 mg/ml collagenase (C0130, Sigma Aldrich) solution to incubate at 37°C for 1 h. It was centrifuged at 1000 rpm for 3 min and resuspended.
  • the rabbit CECs were seeded at the density of 8 x 10 4 /cm 2 and cultured on the gelNF membrane until they became confluent and prepared into a circular shape with a diameter of 8 mm using a biopsy punch.
  • an incision was made using a 2.5 mm (8065921501, Alcon) knife, followed by injection into the anterior chamber using a glass Geuder (CorneaGen). Subsequently, an air bubble was introduced using a cannula and allowed to wait for 5 minutes. After removing the air bubble, cell culture medium was added. After 3 days of incubation, the cells and gelNF membrane were examined using a confocal microscope. [0081] Immunofluorescence staining.
  • the samples were rinsed with Phosphate-Buffered Saline (PBS, Thermo Fisher Scientific) and fixed using 4% paraformaldehyde (15710, Electron Microscopy Sciences) for 30 min at RT followed by 3 times wash using PBS.
  • the samples were then permeabilized using 0.2% triton X-100 (93443, Sigma Aldrich) for 5 min followed by 3 times washing with PBS.
  • the samples were subsequently incubated with 3% Bovine serum albumin (BSA, A2153, Sigma Aldrich) dissolved in PBS for 1 h at RT, then incubated primary antibody dissolved in 1% BSA solution for overnight at 4°C.
  • BSA Bovine serum albumin
  • the primary and secondary antibodies used in this study are ZO-1(339188, Thermo Fisher Scientific), Na + /K + -ATPase (sc- 48345, Santa Cruz Biotechnology), N-cadherin (ab98952, Abcam), Aquaporin-1(ab219055, Abcam), phalloidin-555 (ab176756, Abcam), and DAPI (62248, Thermo Fisher Scientific).
  • the text was visible through the membrane, however, it was more clearly visible with the 3, 5, and 10 min spun gelNF membranes compared to the 30 and 120 min spun ones (FIG.1A).
  • the thickness measured with a Vernier caliper, increased proportionally with spinning time. Specifically, the thickness was approximately 150 ⁇ m for 120 min, less than 20 ⁇ m for 10 min, and less than 10 ⁇ m for 5 min spinning times.
  • the sample subjected to 5 minutes of spinning and 5 minutes of crosslinking exhibited better shape maintenance in the liquid, in contrast to the sample spun for 3 minutes, which displayed a contracted shape (FIG.1A).
  • IhCEC culture on top of the gelNF membrane The cytotoxicity assessment of the gelNF membrane through the Live & Dead assay on IhCEC demonstrated predominant cell attachment in a viable state on both day 3 and 7 (FIG.7A and B). The calculated live cell ratio indicated that 93% and 97% of IhCECs remained viable on days 3 and 7, respectively (FIG. 7C). Following the verification of suitable characteristics of gelNF as a corneal graft material, CECs were cultured to assess cellular compatibility. IhCECs cultured in gelNF showed comparable proliferation to TCP after 24 hours (FIG. 4A), and it was confirmed that high- density culture was possible up to day 7 (FIG. 4B).
  • PrCECs were observed to proliferate initially, and from day 3, they became tightly packed with a decreased elongation ratio, forming a normal hexagonal shape and junctional proteins (FIG.8B-D). Furthermore, when PrCECs were cultured in gelNF for one day, phase-contrast imaging revealed the absence of detached cells, and Live & Dead staining indicated that most cells were viable (FIG. 5A). Immunofluorescence staining for functional protein expression showed that, comparable to the control FNC coating mix-coated cover slips, gelNF gradually formed tight junctions from day 3, becoming more pronounced by day 7. Adherent junctional proteins and aquaporin-1 were also expressed in a similar pattern to the cover slip substrate (FIG.5B).
  • FIG. 6B Following 5-minutes of air bubble incubation, stable attachment of the membrane to the cornea after addition of culture media was confirmed (FIG. 6B). Throughout a 3-day culture period and subsequent fixation and washing steps, the membrane remained firmly adhered to the cornea (FIG.6C). Immunofluorescence staining of phalloidin, ZO-1, and nuclei in the transplanted CECs cultured gelNF membrane, demonstrated a lower density of stromal cells in the cornea with DM removed. Conversely, in the region where the gelNF membrane was transplanted, a higher density of CECs (nuclei and phalloidin) were confirmed. (FIG.6D and E).
  • the gelNF membrane exhibited autofluorescence, allowing the confirmation of ZO-1 expression (white arrows) through partial z-stacked imaging (FIG.6F).
  • the primary role of corneal endothelium is to maintain cornea clarity and thickness by regulating the movement of nutrients and water through the cornea via cell-cell tight junctions and the action of the Na + /K + -ATPase pump. To preserve this critical function, any material used for CEC transplantation must be permeable.
  • the aim of our research is to design a carrier for cultivated CECs that optimizes transparency and thus visual acuity without reducing efficient permeability, a vital aspect of CEC functionality.
  • GA crosslinking is an efficient crosslinking method for electrospun gelNF membrane with a short crosslinking time.
  • studies utilizing the GA vapor crosslink system exposure for several hours did not pose toxicity issues.
  • a process was carried out to remove residual reagent over a sufficient period, suggesting a reduced risk of cytotoxicity (FIG.4A, 5A, and FIG.7).
  • Gelatin is a substance derived from collagen and is utilized in various research fields due to its high biocompatibility. There have been studies on producing nanofibers using electrospinning, mostly blending them with synthetic polymers to enhance mechanical properties.
  • the mechanical property of the endothelium graft is required be appropriate to endure the stretching forces and maintain integrity during surgery and post-transplantation as the cornea goes through various movements and stresses. Moreover, an optimal balance of tensile strength is necessary to ensure that the material is strong enough to maintain its structural integrity and support the endothelial cells, while also allowing for necessary flexibility and compliance within the corneal environment.
  • the gelNF membrane demonstrated suitable properties for transplantation (FIG.2B and FIG. 6A) and appropriate degradation rates (FIG. 3C), addressing the limitations of materials that degrade too rapidly during cell cultivation while maintaining desired properties for CEC delivery.
  • the thickness is one of the crucial considerations for the material intended for transplantation into the corneal endothelium.
  • the gelNF membrane used in this study was optimized at less than 20 ⁇ m after 5 minutes of spinning. Considering that carriers utilized for DMEK should be less than 50 ⁇ m, the gelNF membrane fabricated in this study is adequate for use as a CEC transplantation material.
  • the other advantages of an electrospun nanofiber membrane are its ability to ensure permeability through pore structures, as reported through various studies. In this study, the porosity of the gelNF membrane produced can be observed through the pore structures between nanofibers in the top view image obtained via SEM (FIG.2A). Additionally, a porous structure can be confirmed through the cross-sectioned view image (FIG.3C, day 0).
  • the permeability test using Dextran is an analytical method primarily utilized in membranes that function as barriers.
  • permeability experiments were conducted using FITC-labeled dextran to ascertain the extent to which actual molecules penetrate the gelNF membrane.
  • both 10 kDa and 70 kDa dextran were observed to permeate the gelNF membrane, indicating its permeability property (FIG. 3A).
  • Permeability was also associated with degradation.
  • a significant difference in permeability was noted seven days after incubating the membrane at body temperature (FIG. 3B).
  • Analysis of the thickness changes in the gelNF membrane revealed that over 88% of the membrane had degraded after incubation at body temperature for 14 days (FIG.3C).
  • the rapid degradation of the gelNF membrane may be advantageous in terms of eliminating potential factors that can induce an inflammatory response within the eye. It is presumed that CECs would have begun migrating from the gelNF membrane to the cornea, considering that the gelNF membrane had already attached to the posterior cornea after 5 minutes of incubation following graft transplantation (FIG.6B). [0091] To maintain the thickness and transparency of the cornea, preserving the pump function of CECs is crucial. To achieve this, it is important to maintain a high cell density of CECs while forming tight junctions. In this study, two types of CECs were tested. Firstly, it was confirmed that gelNF membrane allows for high cell density cultivation of IhCECs without cytotoxicity (FIG. 4A and B).
  • the artificial anterior chamber considered an excellent tool for DMEK surgery practice, was utilized in this experiment to assess the material prior to transplantation in an animal model. Grafts inserted into the anterior chamber through a glass geuder cannula were affixed to the posterior of the cornea using an air bubble for only 5- minutes (FIG.6B). This attachment occurred much faster than the several hours or days of gas bubble use with supine positioning in current DMEK procedures. While the reasons for this relatively rapid adhesion are not fully known, it is likely a result of high surface contact area as a result of the mesh-like surface topology of the gelatin nanofibers.
  • the reduced adhesion time presents an advantage of reduced patient discomfort with strict supine positioning in the post-operative period and eliminating the need for re-bubbling procedures.
  • Subsequent examination of cytoskeleton (phalloidin) and tight junctional protein expression in CECs post-transplantation revealed sustained expression for three days when in contact with both the gelNF membrane and the cornea (FIG.6E and F).
  • the clear visualization of ZO-1 expression was aided by employing partial z-stack imaging to confirm tight junctional protein formation (FIG.6F, white arrows).
  • an engraftment test was conducted using rabbit corneas over a period of three days.
  • corneal endothelial cells migrated to the decellularized cornea within three days of cultivation. Consequently, it is anticipated that, during the 14-day degradation period of the membrane, there is ample time for corneal endothelial cells to migrate to the cornea. To validate this accurately, transplantation results need confirmation through subsequent studies. However, considering that the membrane adhered to the cornea during the washing and fixation processes after three days of cultivation, it is believed that interactions may have occurred between cells and corneal tissue or between the gelNF membrane and corneal tissue. [0093] The objective of this study was to develop a material for transplanting cultured CECs. A gelNF membrane was developed using electrospinning that maintained sufficient thickness, transparency, and permeability for proper corneal functionality. We also confirmed that CECs expressed functional proteins effectively.

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Abstract

L'invention concerne des membranes de nanofibres électrofilées. Les membranes sont électrofilées à partir d'un biopolymère biocompatible et biodégradable approprié, par exemple de la gélatine, du collagène, etc., pour générer une membrane, laquelle est ensuite réticulée. La membrane peut être ensemencée avec des cellules. Les compositions trouvent une utilisation dans la régénération tissulaire, en particulier dans la réparation, la régénération et/ou la reconstruction de défauts lamellaires ou partiels de tissu cornéen lésé.
PCT/US2024/061438 2023-12-22 2024-12-20 Membrane de nanofibres électrofilées pour transplantation de cellules cornéennes Pending WO2025137557A1 (fr)

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CN120478308A (zh) * 2025-07-17 2025-08-15 乐比(广州)健康产业有限公司 一种基于天然提取物的护眼组合物及其制备工艺

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US20190269826A1 (en) * 2014-05-12 2019-09-05 Gholam A. Peyman Method Of Corneal Transplantation Or Corneal Inlay Implantation With Cross-Linking
US20220185926A1 (en) * 2020-12-11 2022-06-16 Te Bios Co., Ltd Scaffold For Artificial Organ Using Acrylic Synthetic Polymer And Preparation Method Thereof
WO2022139573A1 (fr) * 2020-12-23 2022-06-30 Universiti Kebangsaan Malaysia Dispositif de cicatrisation cornéenne et son procédé de production

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US20190269826A1 (en) * 2014-05-12 2019-09-05 Gholam A. Peyman Method Of Corneal Transplantation Or Corneal Inlay Implantation With Cross-Linking
US20220185926A1 (en) * 2020-12-11 2022-06-16 Te Bios Co., Ltd Scaffold For Artificial Organ Using Acrylic Synthetic Polymer And Preparation Method Thereof
WO2022139573A1 (fr) * 2020-12-23 2022-06-30 Universiti Kebangsaan Malaysia Dispositif de cicatrisation cornéenne et son procédé de production

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Publication number Priority date Publication date Assignee Title
CN120478308A (zh) * 2025-07-17 2025-08-15 乐比(广州)健康产业有限公司 一种基于天然提取物的护眼组合物及其制备工艺
CN120478308B (zh) * 2025-07-17 2025-09-09 乐比(广州)健康产业有限公司 一种基于天然提取物的护眼组合物及其制备工艺

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