[go: up one dir, main page]

WO2025099175A1 - Matériau composite vivant pour produits médicaux - Google Patents

Matériau composite vivant pour produits médicaux Download PDF

Info

Publication number
WO2025099175A1
WO2025099175A1 PCT/EP2024/081545 EP2024081545W WO2025099175A1 WO 2025099175 A1 WO2025099175 A1 WO 2025099175A1 EP 2024081545 W EP2024081545 W EP 2024081545W WO 2025099175 A1 WO2025099175 A1 WO 2025099175A1
Authority
WO
WIPO (PCT)
Prior art keywords
composite material
pva
inm
hydrogel
pinocembrin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/081545
Other languages
German (de)
English (en)
Inventor
Maria Aránzazu DEL CAMPO BÉCARES
Shrikrishnan SANKARAN
María PUERTAS BARTOLOMÉ
Florian RIEDEL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inm Leibniz Institut Fuer Neue Materialien Gemeinnuetzige GmbH
Original Assignee
Inm Leibniz Institut Fuer Neue Materialien Gemeinnuetzige GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE102023131356.8A external-priority patent/DE102023131356A1/de
Application filed by Inm Leibniz Institut Fuer Neue Materialien Gemeinnuetzige GmbH filed Critical Inm Leibniz Institut Fuer Neue Materialien Gemeinnuetzige GmbH
Publication of WO2025099175A1 publication Critical patent/WO2025099175A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/04—Dispersions; Emulsions
    • A61K8/042—Gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/81—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions involving only carbon-to-carbon unsaturated bonds
    • A61K8/8129—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal or ketal radical; Compositions of hydrolysed polymers or esters of unsaturated alcohols with saturated carboxylic acids; Compositions of derivatives of such polymers, e.g. polyvinylmethylether
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9706—Algae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728—Fungi, e.g. yeasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • 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
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • 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
    • A61L27/52—Hydrogels or hydrocolloids
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow

Definitions

  • the invention relates to composite materials for medical pro- products, medical products with this composite material, processes for its production and use, as well as a Matrix material for composite materials.
  • Hydrogels are three-dimensional networks of cross-linked hydro- philic polymers or fibrillar structures, which have a high Contain a certain amount of water. Such materials are known as matrix materials for biological applications such as drug delivery, wound materials, tissue engineering, and can also be used in cell culture. Their aqueous and porous structure allows for good transport of nutrients to the cells.
  • hydrogels for example, collagen, Gelatin, polyvinyl alcohols or polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • INM-426 INMPT22046DEWO 11/07/2024 Different reactions and mechanisms were investigated for crosslinking the hydrogels, for example photopolymerization, micro- chae addition or similar. This also includes a physical Crosslinking is possible, in which the polymers or fibrils are linked by physical interaction without or in addition to covalent Form bonds in a network.
  • Hydrogels themselves are known as matrices for cell therapies. However, the hydrogels serve as temporary carriers matrix and are mostly used only for eukaryotic cells.
  • Hydrogels are also used as carrier materials for probiotic applications, although these dissolve during use to release the embedded probiotic cells.
  • Other applications of hydrogels include materials for cartilage replacement.
  • lubricity is of great importance and determines the performance and durability of the implant.
  • Hydrogels are also used as coatings for medical devices to increase biocompatibility with tissue. Here, low adhesion to tissue is important to avoid tissue damage.
  • the deposition of proteins and other biomolecules on the surface of the hydrogel reduces lubricity and limits the long-term performance of the devices.
  • hydrogels are also used as a depot for antibiotics or lubricants. For this purpose, these compounds are previously stored in the hydrogel.
  • INM-426 INMPT22046DEWO 11/07/2024 The limited capacity of the hydrogel to absorb hydrophilic molecules by soaking and the rapid excretion of the However, components in the physiological environment limit the Duration of effect, for example in wetting and lubricating effect, for several hours.
  • the incorporation of such lubricants or antifouling agents can also inhibit the growth of a correspondingly coated surface (antifouling).
  • these coatings have only limited regeneration capabilities, for example, when the internal reservoir of the antifouling agent is empty.
  • the object of the invention is to provide a material which allows the easy delivery of substances and which which overcomes the disadvantages of the prior art. Solution This problem is solved by the inventions having the features of independent claims resolved.
  • a composite material for medical nic products comprising at least one matrix material and INM-426 INMPT22046DEWO 11/07/2024 at least one microorganism, wherein the at least one microorganism can release at least one substance into the environment of the composite material.
  • the matrix material is preferably a porous material in which ches microorganisms are embedded. The microorganisms are capable of producing at least one substance to the environment of the composite material.
  • these contain at least one substance from the microorganisms through the matrix material into the environment of the composite material, particularly by diffusion through the matrix material.
  • the matrix material is compatible with the microorganisms during processing and/or use of the composite material.
  • the matrix material allows the diffusion of water, nutrients, and gases to the microorganisms.
  • the matrix preferentially allows proliferation, i.e., growth and proliferation of cells, prefers growth. In particular the control of growth.
  • the composite material retains the microorganisms for at least a certain period of time, preferably for at least 5, 6, 9, 10, 15, 20 or 25 days. Periods of several days and years are also possible.
  • the matrix material embeds the microorganisms. This can be done, for example, via a fiber structure, in cavities, or embedded in cross-linked matrix material.
  • the matrix material can consist of one or more components especially if it is a cross-linked matrix material.
  • the composite material retains moisture and/or is hydrated (water absorption) to ensure the survival of microorganisms. This allows the matrix material to create an environment which allows the survival of the microorganisms. This can include water absorption, the presence of nutrient salts, buffers, or other substances.
  • the matrix material can be a continuous material (e.g. hydrogel) or it may have an internal morphology (porous matrix, fibrillar matrix such as electrospun fabric).
  • the composite material can be structured, e.g., a multilayer film or a printed multicomponent scaffold.
  • the composite material can comprise multiple layers. These can be arranged above, below, and/or within the areas containing matrix material and microorganisms.
  • the composite material can, in principle, have any shape, depending on the application: spherical, planar, three-dimensional, of any geometry and size.
  • the composite material can be one-dimensional (1D, thread), two-dimensional dimensional (2D, such as supported film or membrane) or three-dimensional dimensional (3D, e.g. printed scaffold).
  • the matrix material can be an inorganic, organic material or an organic-inorganic hybrid material. It can be a natural or a synthetic material.
  • the matrix material comprises a hydrogel, in particular made of synthetic or natural polymers. Examples of natural materials are natural polymers such as alginate, chitin, agarose or gelatin.
  • Examples of synthetic materials are polymers such as poly- acrylates (such as polyhydroxyethyl methacrylate (PHEMA)), PVA (Po- lyvinyl alcohols), PEG (polyethylene glycols), acrylated PEG (PEGDA), dextrans, Pluronic, silicones or the precursor compounds listed polymers or copolymers of the mentioned Polymers, as well as combinations thereof.
  • An example of an inorganic matrix material is silica.
  • INM-426 INMPT22046DEWO 11/07/2024 Particularly preferred is a hydrogel comprising one or more Precursors. This means that the hydrogel is produced from these precursors, preferably by forming crosslinks.
  • Stable or transient network or percolation morpho- logies are formed by chemical (formation of covalent bonds), physical (thermo- or pH-sensitive gelation) or other attractive interactions (H-bonding, electrostatic Interactions, metal-ion or ionic complexation)
  • the formation of covalent bonds can also enzymatically.
  • the hydrogel is particularly preferably a cross-linked hydrogel.
  • the matrix material can be mono- or multi-phase (e.g. block copolymers).
  • the matrix material can be degradable or non-degradable. This means that it can be decomposed by biological processes.
  • the matrix material can be transparent or opaque. The lifetime of the composite material is variable.
  • the matrix material can be cross-linked, for example by adding Addition of appropriate crosslinkers during production.
  • the matrix material is preferably a material based on polyvinyl alcohol (PVA).
  • PVA polyvinyl alcohol
  • Particularly preferred is PVA which is at least partially modified with vinylsulfone groups, which crosslink to form the hydrogel, or have been crosslinked. Vinylsulfone groups preferably react with free hydroxyl groups of the PVA.
  • the PVA can also be partially modified with other groups, such as acetic acid.
  • the precursors have a chemical structure, a morphology, or carry Functionalities (e.g. reactive acrylate groups or vinyl groups) pens), which enable them to survive at body temperature and conditions, network structures or percolated morphologies
  • crosslinking can also occur through external influences, such as temperature or electromagnetic radiation. This can depend on the chemistry of the crosslinking.
  • the composite material can also comprise mixtures of several polymers. or polymers with the same backbone, but different mass and/or modification. INM-426 INMPT22046DEWO 11/07/2024
  • the composite material can also comprise mixtures of several polymers or polymers with the same backbone but different mass and/or modification.
  • the matrix material can be a mixture of crosslinked polymer and uncrosslinked polymer, such as crosslinked polyvinyl alcohol and polyvinyl alcohol. A proportion of uncrosslinked polymer of 0 to 20% is preferred. The uncrosslinked polymer can be released after the hydrogel has formed, creating gaps for the microorganisms. It may therefore be necessary to wash the composite material accordingly before use.
  • the high proportion of cross-linked polyvinyl alcohol increases the stability of the hydrogel.
  • a polyvinyl alcohol is used which is modified to a proportion of 2 to 8%, preferably 2 to 6%. are particularly preferably with vinylsulfone groups.
  • the proportion refers to the number of modified hydroxyl groups of the backbone relative to the number of repeating units in the polyvinyl alcohol, which are defined as follows –(- CH 2 -CHOH-CH 2 -CHOH-), where the OH groups can be modified or unmodified.
  • the corresponding monomer is also shown in Figure 4 a).
  • the matrix material is preferably soluble in water.
  • the matrix material is preferably soluble in water to an extent of up to 15 wt. %, preferably to a proportion of 2 to 15 wt. %.
  • the composite material without microorganisms has a water content of at least 50%, particularly preferably at least 60%, most preferably at least 70%, in particular from 70% to 90% (measured as EWC).
  • the composite material a refractive index between 1.31 and 1.37 (at 550 nm).
  • the composite material or precursors thereof can be used in various forms by casting, centrifugal casting, electrospraying, electrospinning, extrusion, stereolithography, laser cutting, Casting, 3D printing, etc. or combinations thereof. the.
  • the composite material (matrix material) is in water, physiological logical buffers or body fluids (e.g. tears, blood, urine, lymph, sweat).
  • the composite material can be applied to the body surface (skin, eye, nails, hair) or placed in the body or in- implanted, e.g., in body organs or on the mucous membranes skin of epithelial tissues (intestine, lungs, internal organs).
  • the composite material can have outer layers for control, Support and/or protect the microorganisms
  • the layers within the composite material can be firmly or liquid. These layers can also have a selective Enable diffusion of the released compound and its fixation on the surface.
  • INM-426 INMPT22046DEWO 11/07/2024 The composite material can be part of another device with additional functions (e.g., as a coating).
  • the composite material can have free or selective diffusion properties.
  • the composite material can allow or prevent oxygen from passing through.
  • the composite material can contain additional molecules to support tion of microorganisms (nutrients, oxygen releasing molecules). These substances are preferably added to the positive material is added from the outside. This can be done continuously, once, or periodically.
  • the composite material can create other parts with additional functions.
  • the at least one microorganism may be a bacterium, a Yeast, a fungus, an algae or a eukaryote or a co-culture of these organisms, preferably a bacterium or a eukaryote.
  • At least one microorganism is the microorganism selected from the group consisting of bacteria, yeasts, filamentous fungi, cyanobacillus teria and microalgae. INM-426 INMPT22046DEWO 11/07/2024 In a further preferred embodiment, the microorganism is selected from the group consisting of Bacillus bacteria teria (e.g.
  • Bacillus subtilis Bacillus megaterium, strep- Streptococcus pyogenes, Streptococcus equi, Streptococcus equisimilis, Streptococcus dysgalactiae, Streptococcus zooepidemicus), Acinetobacter bacteria, Norcardia baceteria, Xanthobacter bacteria, Escherichia bacteria (e.g. E. coli (e.g. strains DH10B, Stbl2, DH5-alpha, DB3, DB3.1, DB4, DB5, JDP682 and ccdA-over (e.g., U.S. Application No.
  • E. coli e.g. strains DH10B, Stbl2, DH5-alpha, DB3, DB3.1, DB4, DB5, JDP682 and ccdA-over (e.g., U.S. Application No.
  • Streptomyces bacteria Erwinia bacteria, Klebsiella bacteria, Serratia bacteria (e.g. S. marcescens), Pseudomonas bacteria (e.g. P. aeruginosa, P. putida), Salmonella bacteria (e.g., S. typhimurium, S. typhi), Megasphaera bacteria (e.g., Megasphaera elsdenii), photosynthetic bacteria (e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus), Chloronema bacteria (e.g., C.
  • green sulfur bacteria e.g., Chlorobium bacteria (e.g., C. limicola)
  • Pelodictyon bacteria e.g., P. luteolum
  • purple sulfur bacteria e.g., Chromatium bacteria (e.g., C. okenii)
  • purple non-sulfur bacteria e.g., Rhodospirillum bacteria (e.g., R. rubrum)
  • Rhodobacter bacteria e.g., R. sphaeroides, R. capsulatus
  • Rhodomicrobium bacteria e.g., R.
  • Corynebacterium bacteria e.g., Corynebacterium glutamicum ATCC 13032, Corynebacterium mastitidis
  • Amycolatopsis bacteria e.g., Amycolatopsis sp. ATCC 39116
  • Corynebacterium glutamicum ATCC 13032 is also known as Corynebacterium glutamicum DSMZ 20300.
  • the microorganism is selected from the group consisting of Yarrowia yeast (e.g. BY lipolytica (formerly classified as Candida lipolytica) adorned)), Candida yeast (e.g. C. revkaufi, C. pulcherrima, C. tropicalis, C.
  • Rhodotorula yeast e.g., R. glutinus, INM-426 INMPT22046DEWO 11/07/2024 R. graminis
  • Rhodosporidium yeast e.g. R. toruloides
  • Saccharomyces yeast e.g. S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis
  • Cryptococcus yeast e.g. T. pullans, T. cutaneum
  • Pichia yeast e.g. P. pastoris
  • Lipomyces yeast e.g. L.starkeyii, L. lipoferus
  • the microorganism Organism selected from the group consisting of Aspergillus fungi (e.g., A. parasiticus, A. nidulans), Thraustochytrium fungi, Schizochytrium fungi and Rhizopus fungi (e.g., R. arrhizus, R. oryzae, R. nigricans), e.g., an A. parasiticus Strain such as strain ATCC24690 or an A. nidulans strain such as the strain ATCC38163.
  • the at least one microorganism is a bacterium particularly preferably a gram-positive bacterium.
  • the matrix according to the invention is capable of cultivating many microorganisms and controlling their growth.
  • the bacterium is a bacterium of the family Corynebacteriumceae, particularly preferably of the genus Corynebacterium. for example Corynebacterium glutamicum. It can also be a bacterium of the Actinobacteria, especially of the family Streptomycetaceae, especially of the genus Streptomyces.
  • the at least one microorganism can be present in the composite material be homogeneously distributed or localized or compartmentalized at a specific location within the material.
  • the microorganism preferably has no direct contact with the environment of the composite material. It is shielded by the matrix.
  • the composite material has an edge region that contains no microorganisms. This outer region can, for example, prevent the diffusion of the microorganisms out of the composite material. For this purpose, it can also, for example, have a higher degree of cross-linking, so that the at least one microorganism is enclosed.
  • the composite material can also be processed in the form of multilayers or core-shell designs.
  • the microorganisms release molecules with biological activity. The molecules can be produced by the microorganisms and/or produced by converting a precursor absorbed by the microorganisms.
  • the at least one microorganism can be evolutionary or genetically nically/metabolically altered.
  • the at least one microorganism can preferentially consume nutrients from the environment of the composite material, light, oxygen and/or absorb CO2.
  • the matrix material can also be degraded and can be a source of nutrients. Nutrients must also be incorporated into the composite material to enable the microorganisms to survive for a certain period of time.
  • INM-426 INMPT22046DEWO 11/07/2024 Preferably, the at least one microorganism is a microorganism organism that can withstand extreme conditions (extremo- phile).
  • the at least one microorganism can substance on site (i.e.
  • the nutrients for the at least one microorganism can be immediately available or provided on demand.
  • the at least one microorganism can be a wild-type or genetically modified organism. be technically/metabolically altered.
  • the composite material can be applied using various methods and in different geometries.
  • the composite material is preferably produced by mixing pre- runner connections and in situ networking through physical, chemical or enzymatic cross-links are produced.
  • the composite material can also be produced by electrospinning poly- melts or polymer solutions.
  • the micro- Roorganisms can be contained in the fibers or captured by the deposited fibers.
  • the composite material can also be processed by electrospraying This may contain microorganisms.
  • the composite material can also be produced by other processes such as Emulsification, microfluidic encapsulation, injection into capsules, bioprinting.
  • INM-426 INMPT22046DEWO 11/07/2024 The substances released can have different properties They can, for example, be regenerative, therapeutic, protective, lubricating, anti-infective, have a probiotic or dietary effect. These are preferably substances that directly or indirectly cause a change in the environment of the composite material. Examples of direct changes include local therapeutic effects, such as disinfection, anti-inflammatory effects, or lubrication. Examples of indirect changes include the release of therapeutically active substances, which only acquire their effect through metabolism.
  • the substance can be produced by the microorganisms continuously, produced on demand, after stimulus or time-dependently.
  • the substance can have a small or large molecular weight ben.
  • the substance can be used in its active form or in a precursor ferform. For example, it may contain a degradable part for enzymatic activation.
  • the composite material can be used especially for coatings with antimicrobial, lubricating, self-protective and therapeutic tical properties can be used.
  • the composite material is for medical products; it is preferably part of a medical product. INM-426 INMPT22046DEWO 11/07/2024 These can be all types of products or medical devices used in the medical field. Examples include coatings for all types of medical devices.
  • Composite material can be used to achieve corresponding sub- punches are released locally, for example to prevent infections to prevent.
  • ophthalmic products such as contact lenses, intraocular lenses, and intraocular implants. In contact lenses, the released substance is not hyaluronic acid. It is also possible to use the composite material to convert substances. For example, substances present in the body or administered externally can be converted into therapeutically active substances by the composite material.
  • the dosage can be controlled.
  • the substance can be administered more easily, for example orally.
  • the composite material can also be used as a coating of bioelectronic devices or sensors whose performance may be affected by biofouling. INM-426 INMPT22046DEWO 11/07/2024 It is particularly suitable for devices that are used in an aqueous stored in a dry environment.
  • the composite material can also be used for health monitoring devices. monitoring or administering medication.
  • the invention also relates to a medical product comprising the composite material according to the invention, preferably medical products as described above.
  • the composite material according to the invention preferably forms only a partial region of the medical product, such as the surface or a defined compartment. If the medical product comprises hydrogels, the composite material according to the invention preferably forms at least a partial region of the hydrogel.
  • the composite material as such or in the medical device can be in the hydrated form as a hydrogel or in a reconstitutable form such as freeze-dried.
  • the composite material can be completely contained within the hydrogel or coatings, for example, arranged in a compartment INM-426 INMPT22046DEWO 11/07/2024 be or at least partially the surface of the hydrogel.
  • the microorganisms can also be incorporated into particles made of inventive according to the invention, which is embedded in the Hydrogel are arranged.
  • the remaining part can also consist of common medical hydrogels, such as polyvinyl alcohol (PVA), polyvinyl acetate (PVAc), poly(meth)acrylates, in particular poly(2-hydroxyethyl) methacrylate (poly-HEMA), polyethylene glycol, polysiloxane, or copolymers of these materials.
  • PVA polyvinyl alcohol
  • PVAc polyvinyl acetate
  • poly(meth)acrylates in particular poly(2-hydroxyethyl) methacrylate (poly-HEMA), polyethylene glycol, polysiloxane, or copolymers of these materials.
  • poly-HEMA poly(2-hydroxyethyl) methacrylate
  • polyethylene glycol polysiloxane, or copolymers of these materials.
  • Acofilcon A Acofilcon B, Alfafilcon A, Altraficon A, Atlafilcon A, Balafilcon A, Bufilcon A, Comfilcon A, Crofilcon, Deltafilcon A, Dimefilcon A, Droxifilcon A, Efrofilcon A, Enfilcon, Epsifilcon A, Etafilcon A, Focofilcon A, Galyfilcon A, Heflicon A, Heflicon B, Hefilcon C, Hilafilcon A, Hilafilcon B, Hi-oxifilcon A, Hioxifilcon B, Hioxifilcon D, Isofilcon, Lidofil-con A, Lidofilcon B, Lotrafilcon A, Lotrafilcon B, Mafilcon, Methafilcon A, Methafilcon B, Narafilcon B, Nelfilcon A, Nesco-filcon A, Netrafilcon A, Ocufilcon A, Ocufilcon B, Ocufilcon C, Ocufilcon D
  • the released substance can therefore be used according to the be adapted.
  • an anti-fouling layer this can be achieved, for example, by forming a surface layer of INM-426 INMPT22046DEWO 11/07/2024
  • the released substance prevents the adhesion of organisms or the deposition of growth molecules.
  • the chemical composition of the released natural antifouling agent may also include antimicrobial (e.g., charged polymers with quaternary amines) or cell-repellent properties. Examples of such a substance are hyaluronic onic acid or lipid compounds.
  • the porous matrix material allows water and nutrients to reach the microorganisms, allowing the antifouling layer to be regenerated at any time. This creates a regenerable antifouling layer.
  • Substances with a hydrating effect are also possible by substances that lead to water retention.
  • the substance can be endogenous or exogenous.
  • the substance can be a chemical molecule, oligomer, polymer, peptide, or protein. It may be a therapeutically active substance. This makes it possible for the therapeutically active substance to be delivered directly to or into the body.
  • These are preferably substances that directly or indirectly cause a change in the environment of the composite material. Examples of direct changes include therapeutic effects, such as the treatment of diseases, disinfection, and inflammation. inhibition, fouling inhibition or wetting or lubrication ability.
  • INM-426 INMPT22046DEWO 11/07/2024 An example of indirect changes is the release of therapeutically active substances, which only acquire their effect through metabolism.
  • substances with therapeutic effects are selected from the group consisting of the following substances: anti-infectives, including, without limitation, antibiotics, antiviral agents, and antifungals; antiallergic agents and mast cell stabilizers; steroidal and non-steroidal anti-inflammatory agents; cyclooxygenase inhibitors, including including, without limitation, Cox-I and Cox-II inhibitors; combination economies of anti-infective and anti-inflammatory agents; decongestants; anti-glaucoma agents, including, but not limited to adrenergics, ⁇ -adrenergic blockers, ⁇ - Adrenergic agonists, parasympathomimetics, cholinesterase inhibitors, carbonic anhydrase inhibitors, and prostaglandins; combinations of anti-glaucoma agents; antioxidants; dietary supplements; drugs for the treatment of cystoid macular edema, including but not limited to non-steroidal anti-inflammatory drugs; drugs for the treatment of
  • antibiotics include aranciamycin or griseorhodines such as griseorhodine A.
  • Substances with positive therapeutic effects can also genes, such as cinnamic acid, vitamins, flavonoids, antioxidants, or derivatives thereof.
  • the substance is a wetting or Lubricant or a lipid, particularly preferably hyaluronic acid.
  • the at least one microorganism then comprises a hyaluronic acid synthetase, such as hasA (hyaluronic acid synthase) from Streptomyces equisimilis, which, for example, a dehydrogenase, such as hasB (ugdA1, dehydrogenase) of Corynebacterium glutamicum.
  • the composite material enables and controls the growth of the microorganisms within the matrix material.
  • the amount and duration of the release can be influenced by different parameters can be influenced. Examples include the concentration of microorganisms in the matrix material, diffusion properties properties of the matrix material and/or encapsulation or coating of the composite material.
  • microorganisms it is also possible to influence the release through the feeding condition or how certain sensors in the microorganism are controlled, as in the case of for example by external stimuli such as light, pathogens, pH value or the temperature. It is also possible for the microorganisms to be regenerated, for example, by storing the composite material in a feeding solution. Alternatively, the microorganisms can also INM-426 INMPT22046DEWO 11/07/2024 be regenerated by the conditions of their environment, for example by absorbing substances from body fluids.
  • the composite material according to the invention is preferably obtained by producing the matrix material in the presence of microorganisms For this purpose, appropriate precursor compounds are preferably reacted with one another.
  • the invention also relates to a process for the preparation a composite material, comprising the following steps: a) providing a composition comprising at least a precursor compound for the matrix material and at least one microorganism; b) reaction of the precursor compounds to form a composite material.
  • Water-soluble precursor compounds are preferred. This indicates that the precursors are necessary under the conditions conditions of the reaction in solution.
  • Particularly preferred are precursor compounds with a mole- molecular weight of over 3000 u. Monomers with such molecular weights usually have lower toxicity to microorganisms, so that the matrix material can be produced in the presence of microorganisms.
  • the precursor compounds are preferably non-toxic to the Cells, ideally precursors with a molecular weight above 3000 u.
  • precursor compounds are polyvinyl alcohols Polyacrylates (PVA), polyacrylates, polymethacrylates such as 2-hydroxyethyl methylacrylate (HEMA), glycerol methacrylate, polyethylene glycol, silicone hydrogels, and copolymers thereof.
  • PVA polyvinyl alcohols
  • polyacrylates polyacrylates
  • polymethacrylates such as 2-hydroxyethyl methylacrylate (HEMA), glycerol methacrylate, polyethylene glycol, silicone hydrogels, and copolymers thereof.
  • the polymers or copolymers may also be modified with reactive or hydrophilic groups, such as acrylic groups, methacrylic groups, carboxylic acid groups, amino groups, hydroxyl groups, Vinyl groups or vinyl sulfone groups.
  • At least one of the precursor compounds is preferred, provided several are present, based on oligomers or polymers.
  • artificial oligomers or polymers are Poly(meth)acrylates, such as poly(meth)acrylamides, poly(meth)acrylic acid, PolyHPMA or PolyHEMA, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane (PU), polyvinylpyrrolidone (PVP), polyamides, poly(amidoamines) (PAMAM), polyesters, polylactides, polyglycolic acid (PGA) or poly(lactide-co-glycolide) (PLGA), polyanhydrides, poly(ortho)esters, polyacetals, poloxamers (block copolymers of ethylene oxide (PEG) and propylene oxide (PPG, Pluro- nic), such as PEG-Co-PPG-Co-PEG), poly-2-oxazoline, polypho
  • Polymers based on polyethylene glycol or polyvinyl chloride are preferred. nyl alcohol.
  • the oligomers and polymers are preferably functionalized with the corresponding functional groups.
  • the 1,2- or 1,3-Aminothiol groups preferably by the corresponding amino acids, such as cysteine or homocysteine.
  • Thiide-based means that the corresponding oligomer is made up of at least 80% of its molecular mass from natural or unnatural amino acids.
  • Such oligomers therefore have at least two aminothiol groups, in particular at least two cysteines. Terminal cysteines, which are linked to the oligomer via the carboxyl group, are preferred.
  • the at least partial use of natural polymers also allows the introduction of specifically cleavable sites in the hydrogel, for example by enzymes. It may be necessary for the functional groups to be linked to the oligomer or polymer via a short linker, for example via one or more esters, ethers, carbamates, urethanes, disulfide bridges, or amide bonds. Linkers with a molar mass of less than 5000 Da, preferably less than 1500 Da, more preferably less than 800 Da, in particular less than 500 Da or less than 200 Da are preferred.
  • the type of functional group depends on the type of crosslinking reaction. It can be hydroxyl groups, ester groups, carboxylic acid groups, unsaturated groups such as acrylate groups.
  • the temperature during the reaction of the precursor compounds is preferably between 20 °C and 80 °C, preferably between 20 °C and 60 °C.
  • the reaction of the precursor compounds occurs preferentially via functional groups of the precursor compounds with crosslinking the precursor compounds.
  • the conditions for the reaction depend on the type of precursor compounds or reactive groups. It may be necessary to add further ingredients to the composition Compounds such as radical initiators or crosslinking initiators can be added.
  • the reaction is preferably triggered by an external stimulus. This can be, for example, heating or irradiation with electromagnetic waves.
  • the precursor compounds react preferentially to form a hydrogel.
  • Hydrogel formation means that The crosslinking results in the formation of a hydrogel. Sufficient crosslinking reactions therefore take place. This can be controlled by the type and quantity of components used.
  • the composite material preferably the matrix material, can also nor contain bioactive reagents, especially for the support of microorganisms or in sensory applications.
  • the invention also relates to a matrix material or precursor fer thereof for use in a composite material according to the invention material as described above for the composite material.
  • INM-426 INMPT22046DEWO 11/07/2024 Particularly preferred is a matrix material based on polyvinyl alcohol, which is at least partially coated with vinylsulfone
  • the vinylsulfone-modified monomer preferably forms Polyvinyl alcohol modified with vinylsulfone groups forms the precursor compound, with the vinylsulfone groups reacting to form the matrix material.
  • Fig. 1 A. Scheme of the pMGE encoded in E. coli Nissle 1917 K2-nar plasmid with the PK2 promoter, which regulates the expression sion of the enzymes that convert cinnamic acid to pinocembrin.
  • B, C Analysis of bacterial growth and pinocembrin production in cultures of E. coli Nissle 1917 pMGE-K2-nar in the presence of cinnamic acid at different concentrations (B) and at different time points (C).
  • Fig. 2 A. Confocal fluorescence microscopy images of living the/dead stained bacteria in chemically cross-linked INM-426 INMPT22046DEWO 11/07/2024 PVA-based hydrogels that can be used for up to 14 days axes (scale: 20 ⁇ m; green: living; red: dead). B. Analysis of the volume fraction of bacterial populations within the imaged volumes over 14 days. C . Analysis of cell viability over 14 days. Each symbol represents the values of a single image. The middle lines are means, and the whiskers are standard deviations of values obtained from 3 images at different locations in 3 independent samples.
  • Fig. 3 A. Protocol for the preparation of bacterial PVA-VS- Bilayer ELM films.
  • B. Photo of a bilayer ELM film with bacterial growth over 13 days. Grid size 0.5 cm. Additional images in Fig. g ur 11;
  • the samples correspond to different time points (from 4 h to 21 days; 4 h, 24 h, 7 days, 14 days, 21 days) and the positive control (no hydrogel, Positive Control).
  • the DNA fragment containing the promoter of interest, an EcoRV restriction site, and the three terminators, was synthetically produced and inserted into the pVV.01 plasmid after PCR amplification.
  • the kanamycin resistance cassette is shown in pink and the pUC ORI sequence in gray. The figure was taken from Kufs et al. 2020 taken over. p: promoter; rbs: ribosomal binding site; t: terminator; INM-426 INMPT22046DEWO 11/07/2024 Fig. 9 Analysis of pinocembrin degradation over 48 hours in the Control culture Nissle 1917 and in LB medium; Fig. 10 Agar smear test to check for leakage of ELM films.
  • S1 control ELM films without bacteria
  • S2 ELM films with E. coli Nissle 1917 pMGE-K2-nar, to which mM cinnamic acid was added from day 64
  • S3 ELM films with E. coli Nissle 1917 pMGE-K2-nar, to which mM cinnamic acid was added from day 4, preventing the bacteria from growing in the films.
  • the supernatants were removed every day from the media in which the ELMs were incubated and applied to LB agar plates. where the numbers are indicated. The exit of bacteria and the contamination of the medium is indicated by the growth of a lawn-like colony at this point; Fig.
  • PVA is a clinically approved hydrogel for use in contact lenses and is also used for the encapsulation of probiotics.
  • PVA-VS hydrogels were prepared by polymerizing tion of PVA-VS and PVA solutions using LAP as Photoinitiator. In screening experiments, solutions with 5-10% w/v concentration of PVA-VS (177 kDa) with a VS functionalization degree of 4.6 ⁇ 0.1%, transparent and mechanically stable hydrogels (Table 1). Encapsulation experiments with C. glutamicum showed that the bacterium could grow in these hydrogel compositions.
  • Blends of 10 and 5% w/v PVA-VS/PVA precursors with PVA-VS/PVA ratios of 100:0, 99:1, and 95:5 (Table 1) formed according to rheological experiments ( Figure 5 a)) within of seconds hydrogels by irradiation at 365-480 nm (6 mW/cm2). An irradiation time of 120 s (420 nm, 6 mW/cm2) for gel formation to ensure complete conversion to ensure the VS-mediated cross-linking reaction. At this wavelength, exposure times of 30-300 s at an irradiance of 6-10 mW/cm 2 Compatible with live bacteria.
  • the 10% PVA-VS hydrogel exhibited a shear modulus (G') of 17.9 ⁇ 0.1 kPa, which is close to the G' of commercially available PVA contact lenses (Table 1, Figure 5b).
  • G' shear modulus
  • PVA-VS content or replacing part of the PVA-VS with PVA, softer hydrogels were obtained (Table 1).
  • Other physicochemical properties important for contact lenses were within the range of commercial PVA- INM-426 INMPT22046DEWO 11/07/2024 based contact lenses (Focus® DailiesTM, Alcon), as shown in Table 1.
  • the molecular mass distribution of the eluted polymer was the same as that of the PVA precursor, indicating that only uncrosslinked PVA was released from the hydrogel.
  • the films were tested using a trans-well plate. prepared ( Figure 5 a)). A mixture of proteins with a Molecular weight between 12.3 and 160 kDa was added to the upper well, and the protein concentration collected in the lower well was determined by BCA assay quantified ( Figure 6 d)). The molecular weight of the mixture was analyzed by electrophoresis to monitor the differences in diffusion kinetics as a function of protein size. ( Figure 5 b), Table 2).
  • Proteins with a molecular weight Proteins with a weight ⁇ 25 kDa diffused through the hydrogels within 4 hours. Proteins with a size >25 kDa required 4 hours to diffuse through the hydrogel containing non-crosslinked PVA and 1 to 14 days when the PVA-VS content increased from 5 to 10% w/v.
  • the developed hydrogels exhibited slow diffusion of INM-426 INMPT22046DEWO 11/07/2024 medium to large molecules. The diffusion rate depends on the size of the protein and can be adjusted by adjusting the content and proportion of PVA-VS and PVA in the hydrogel. Methods: The 1H NMR spectra in D2O were recorded with a Bruker Avance 300 MHz.
  • the Tecan Infinite M Plex multimode plate reader was used for transmission, absorption, and bioluminescence measurements.
  • An aqueous gel permeation chromatography/size exclusion chromatography system (Agilent 1260 Infinity II Multi-Detector GPC/SEC) equipped with Ult- violet, refractive index, light scattering and viscosity de- detectors, was used to measure the release of PVA and HA from hydrogel supernatants.
  • Microscopic images were taken using a Zeiss LSM-880 confocal laser scanning microscope (Zeiss). Images were captured using a Zeiss Plan-Apochromat 63x/1.4 Oil DIC M27 objective.
  • the reaction mixture was purified for 3 days by dialysis against Milli-Q water using a Spectra/Por dialysis membrane (MWCO: 3.5 kD, Spectrum Laboratories, USA). After dialysis, the PVA-VS was freeze-dried and stored at room temperature. temperature ( Figure 4 a)).
  • the molecular weight of the purified PVA-VS was quantified by GPC using 0.1 M NaCl and 30% MeOH as the mobile phase at a flow rate of 1 ml/min. The measurements were carried out at 35 °C and 51 bar.
  • a column combination consisting of a PSS SUPREMA LUX pre-column (dimensions 8 x 50 mm) and a PSS SUPREMA LUX pre-column (dimensions 8 x 50 mm) was used. 2 , particle size 10 ⁇ m) and a PSS SUPREMA Linear M (dimensions 8 x 300 mm 2 , particle size 10 ⁇ m) separation column was used.
  • An Agilent RID detector with positive signal polarity was used at 35 °C.
  • PVA-VS and PVA stock solutions INM-426 INMPT22046DEWO 11/07/2024
  • PVA-VS and PVA solutions with a concentration of 10% w/v in water were prepared by heating at 90 °C for 4 hours. Higher polymer concentrations did not result in homogeneous solutions in water, and at concentrations of >5% w/v in BHI, a physical gel formed.
  • Preparation of PVA-VS/PVA hydrogels PVA-VS and PVA stock solutions were combined to form a total polymer concentration concentration of 5% (w/v).
  • PVA-VS/PVA- Ratios (95:5, 99:1 and 100:0) were measured at room temperature by mixing the appropriate volumes of the stock solutions and diluting with a 1% (w/v) solution of the LAP photoinitiator in BHI 2x medium. The concentration of the LAP initiator in the final mixture was 0.5% (w/v).
  • PVA-VS/PVA 100:0 hydrogels (10% w/v) LAP was added directly to the PVA-VS stock solution at a concentration of 0.5% w/v. The precursor solutions were incubated for 10 minutes.
  • EWC Equilibrium water content
  • oxygen permeability The EWC and oxygen permeability of PVA-VS/PVA hydrogel discs and the commercial Focus® DailiesTM contact lenses were evaluated.
  • Optical transmittance (transmission) PVA-VS/PVA hydrogels (40 ⁇ L of the polymer precursor solution) were prepared from the stock solutions and molded into a transparent INM-426 INMPT22046DEWO 11/07/2024 96-well plate with flat bottom by photocrosslinking with a UV light source (420 nm) at an irradiance of 6 mW/cm 2 for 2 minutes. The optical transmittance of the hydrogels was measured using a plate reader.
  • the measurements were performed after hydrogel preparation and after incubation for 2 and 24 hours in STF at 30 °C.
  • the absorbance measurements were performed at wavelengths from 400 to 800 nm in 5 nm steps, and the optical transmittance was calculated from the absorbance measurements.
  • the values at 600 nm are listed in Table 1.
  • the measurements were performed in groups of four, and mean and standard values were Refractive index: PVA-VS/PVA hydrogel discs with a diameter of 12 mm and a height of 300 ⁇ m (37 ⁇ L of the polymer precursor solution) were produced by molding as described above.
  • the pre- prepared hydrogel samples and the commercial Focus® DailiesTM contact lenses were incubated in STF for 24 hours at 30 °C.
  • the refractive index of the hydrated samples was determined using a refractometer. The measurements at 589.3 nm are shown in Table 1 for comparison purposes.
  • PVA release PVA-VS/PVA hydrogel discs with a diameter of 12 mm and a height of 300 ⁇ m (37 ⁇ L polymer precursor solution) were as previously described, prepared by molding and in 1.5 mL STF at 30 °C. After specific time points (3, 5, 7, 14, and 21 days), the supernatants were collected and analyzed using the GPC-SEC system. The injection volume was 20 ⁇ l.
  • INM-426 INMPT22046DEWO 11/07/2024 The mobile phase was 0.2 M NaNO3 (99.995% pure) in deionized tem water and 0.2 M NaNO3 (99.99% pure) in TLC water at a flow rate of 1 mL/min.
  • a PL aquagel-OH MIXED-H 8 ⁇ m, 7.5 x 300 mm SEC column (part number: PL2080-0700) was used. with a particle size of 8 ⁇ m and a molecular weight range of 6-10,000 kDa.
  • PVA-VS/PVA hydrogels in a ratio of 95:5 and 100:0 at 5% w/v and in a ratio of 100:0 at 10% w/v were prepared on the bottom of a Transwell insert (Falcon® Cell Culture Inserts, transparent PET membrane with 8.0 ⁇ m pore size).
  • Transwell insert Falcon® Cell Culture Inserts, transparent PET membrane with 8.0 ⁇ m pore size.
  • 45 ⁇ L of the hydrogel precursor solutions (prepared as described above) were pipetted into the Transwell insert and cross-linked (405 nm, 6 mW/cm 2 , 2 min), resulting in hydrogels with a diameter of 6.3 mm and a thickness of 1 mm.
  • the protein solution was loaded in the same buffer in an equimolar ratio of 5.5 ⁇ M for each protein and with a total protein concentration of 1 mg/ml.
  • the samples were incubated at 30 °C. different incubation times (1, 2, 3, 7, 10, 14, 21 days; Figure 7 a): diffusion), 25 ⁇ l samples were taken from the bottom of each well and DPBS1x buffer added (Figure 7 a) Collection) to determine the total volume of the well changed.
  • the structure is shown in Figure 7 a).
  • Protein quantification was performed using the BCA assay (PierceTM BCA Protein Assay Kit, Thermo ScientificTM) by colorimetric detection with a plate reader (562 nm) of the total protein concentration in the samples using a Calibration line ( Figure 7 a): Detection).
  • the ca- The calibration line was obtained by measuring absorbance at 562 nm of a series of BSA standard solutions (concentration range from 25 ⁇ g/mL to 2000 ⁇ g/mL, prepared in DPBS1x buffer) treated according to the BCA assay kit protocol.
  • An ANOVA was performed using the Tukey grouping method of the total protein content obtained for the different samples. content at a significant level of *p ⁇ 0.05.
  • Flavonoids are a group of natural products synthesized primarily by plants and found in many foods. Their basic chemical structure consists of two benzene rings connected by a heterocyclic pyran ring.
  • flavonoids are being investigated as potential drug candidates for various diseases.
  • the flavonoid pinocembrin has shown therapeutic potential in skin fibrosis and keloid formation in mouse studies (Li et al., 2021).
  • pinocembrin has potentially neuroprotective effects in the treatment of Alzheimer's disease (Liu et al., 2014) and Parkinson's disease (Wang et al., 2014), anti-inflammatory activity (Zhou et al., 2015), antioxidant activity (Shi et al., 2011), antimicrobial activity (e.g., against Staphylococcus aureus) (Soromou et al., 2013), vasodilatory activity (Li et al., 2013), and hepatoprotective activity (Ma et al., 2023).
  • Alzheimer's disease Liu et al., 2014
  • Parkinson's disease Wang et al., 2014
  • anti-inflammatory activity Zhou et al., 2015
  • antioxidant activity Shi et al., 2011
  • antimicrobial activity e.g., against Staphylococcus aureus
  • vasodilatory activity Li et al., 2013
  • hepatoprotective activity Mo e
  • flavonoids such as pinocembrin
  • flavonoids due to a number of factors, it can be challenging One challenge is the limited availability of natural sources, as many flavonoids occur in relatively small amounts in certain plants (Zhou et al., 2014).
  • the chemical synthesis of some flavonoids can also be complex, requiring several steps and increasing the cost and difficulty of production (Selepe and Van Heerden, 2013).
  • some flavonoids are chemically unstable and can easily decompose or degrade (Lu et al., 2019), making their production and storage difficult.
  • a promising alternative for the synthesis of flavonoids is microbial biofactories, which can be easily scaled up (Pandey et al., 2016; Okoye et al., 2023).
  • INMPT22046DEWO 11/07/2024 Significant progress has been made in the identification of biosynthetic pathways for various flavonoids in industrially relevant microbial hosts such as E. coli (Huang et al., 2022), C. glutamicum (Wu et al., 2022), and yeast (Tartik et al., 2023). Enzyme cascades in E. coli BL21 (DE3) were recently developed for the synthesis of various flavonoids (Kufs et al., 2020).
  • flavonoids By expressing a CoA ligase from Nicotiana tabacum and a chalcone synthase from Arabidopsis thaliana, various flavonoids, including pinocembrin, could be produced by adding cinnamic acid derivatives as a substrate.
  • a major challenge in the microbial production of flavonoids is that the production yield must be improved to make this approach economically viable.
  • synthetic production methods cannot economically compete with extraction from natural sources (Tous Mohedano et al., 2023; Sheng et al., 2020; Okoye et al., 2023). Therefore, alternative strategies to improve the availability of such flavonoids for medicinal use are highly desirable.
  • a possible alternative approach to making flavonoids affordable could be to engineer probiotic bacteria to produce them directly in the body. However, this strategy would require introducing genetically modified bacteria into the body, so their biocontamination must be ensured at the specific site of application. Since flavonoids are bioactive compounds, the amount and duration of production must also be controlled.
  • the ELM corresponds to the composite material of the invention. In ELMs, metabolically active microbes are enclosed and held in a material that allows the diffusion of nutrients, oxygen, and metabolites.
  • ELMs are intended as living drug delivery devices for the production and delivery of drugs in the body (Rodrigo-Navarro et al., 2021). Drug delivery can be controlled by introducing genetic switches (Rivera-Tarazona et al., 2021; Dhakane et al., 2023) or by controlling the mechanical and diffusion properties of the embedding matrix (Bhusari et al., 2022; Bhusari et al., 2023; Priks et al., 2020).
  • This example describes an ELM consisting of probiotic bacteria engineered to produce a flavonoid encapsulated in a poly(vinylal- alcohol) (PVA) hydrogel film.
  • PVA poly(vinylal- alcohol) hydrogel film
  • the sequences were selected from the iGem catalog (https://parts.igem.org).
  • the selected constitutive promoters were BBa_K823004 (here referred to as K2), BBa_J45993 (OY), and BBa_J01006 (R10).
  • Three different synthetic DNA elements designed (in Table 5) using the primers pVV.rec.F and pVV.rec.R were amplified and cloned into the basal vector pVV-01 (Hoefgen et al., 2018).
  • each plasmid containing the 4Cl gene was recombined with the corresponding plasmid containing the Chs gene, creating an expression vector containing both genes under the control of the selected constitutive promoter.
  • the resulting plasmids and the The oligonucleotides used are listed in Tables 3 and 4. Culture of E.
  • the cells were resuspended in 0.1 M CaCl 2 resuspended with 20% glycerol and stored on ice or at -80°C until transformation.
  • chemically competent E. coli Nissle 1917 cells were mixed with 100 ng of pMGE-K2-nar plasmid DNA and incubated on ice for 30 minutes. Transformation was performed with a heat shock at 42°C for 30 seconds, followed by cooling on ice for 2 minutes.
  • the cells were resuspended in 950 ⁇ l of LB medium and incubated for 1 hour at 37°C and 180 rpm.
  • LB Kn50 50 ⁇ g/ml kanamycin
  • a 10% PVA-VS solution in water was mixed with the bacterial suspension in LB Kn50 and the lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator solution (LAP, Sigma Aldrich) in 2x LB Kn50 to achieve a final PVA-VS concentration of 5% w/v, a 0.5% w/v concentration of LAP, and a bacterial OD600nm of 0.05 ( ⁇ 4 x 10 6 cells/ml) in the mixture.
  • the hydrogels were prepared by photoinitiated radical crosslinking of the VS groups using a UV light source (365-480 nm) at an irradiance of 6 mW/cm 2 for 2 minutes.
  • ELMs were used in the form INM-426 INMPT22046DEWO 11/07/2024 of bacterial hydrogel films on glass coverslips (13 mm diameter, 16 mm thick). The glass was previously coated with 3-(trimethoxysilyl)propylacrylate to facilitate the covalent bonding of the hydrogel to the glass surface.
  • the films consisted of two layers and were prepared in two steps.
  • Z-stacks of 18.44 ⁇ m were acquired at a Z-step size of 0.45 ⁇ m. Images with a size of 134.95 ⁇ 134.95 ⁇ m (1024 ⁇ 1024 pixels) were acquired with double line averaging and a pixel dwell time of 0.42 ⁇ s. Representative images were acquired with the maximum intensity Z-projection (Zen 2.3 SP1, Zeiss, Oberkochen, Germany). The Imaris surface tool (Imaris v9.8, Bitplane, Zurich, Switzerland) was used to calculate the 3D volume of the live and dead bacteria. The surfaces were generated with a smoothing function of 0.264 ⁇ m, a diameter of the largest sphere of 10 ⁇ m, and an automatic threshold.
  • the volume fraction of live/dead bacterial colonies in the hydrogel samples was calculated as the sum of all volumes of the live/dead colonies divided by the total volume of the imaged region (335820 ⁇ m 3 ).
  • the viable fraction was calculated as the volume of viable colonies divided by the total volume of viable and dead colonies.
  • the bacterial hydrogels were incubated with 400 ⁇ l of LB Kn50 + 4, 2, 0.5, and 0.125 mM.
  • INM-426 INMPT22046DEWO 11/07/2024 Cinnamic acid medium Five technical replicates were prepared for each condition, while one replicate was incubated in LB Kn50 as a control. The supernatant was removed every 24 hours and replaced with appropriate fresh media. 100 ⁇ l of the supernatants were centrifuged in a benchtop centrifuge, transferred to a fresh 1.5 ml Eppendorf tube, and stored at -20 °C until LC-MS measurement.
  • the ELM films were first incubated at 37 °C in a 24-well plate containing 400 ⁇ l of LB Kn50 medium for 3 days to allow the bacteria to grow. Starting on day 4, the films were covered with 400 ⁇ l of LB Kn50 + 4 mM cinnamic acid medium and LB Kn50 as a control and kept at 37°C for 3 days, with the medium being replaced daily. Six technical replicates were prepared for both conditions. The supernatant was removed every 24 hours. and replaced with appropriate fresh media. 100 ⁇ l of the Supernatants were centrifuged in a benchtop centrifuge, transferred to a fresh 1.5 ml Eppendorf tube, and stored at -20°C until LC-MS measurement.
  • LC/ESI QTOF-MS development of an LC/ESI QTOF-MS method for the quantification of pinocembrin in supernatants.
  • the LC/ESI QTOF-MS analysis is performed using a 1260 Infinity LC in combination with a 6545A high-resolution time-of-flight mass analyzer, both from Agilent Technologies (Santa Barbara, CA, USA).
  • the separation of 1 ⁇ l of sample is performed using a Poroshell HPH-C18 column (3.0 x 50 mm, 2.7 ⁇ m) equipped with the same guard column (3.0 x 5 mm, 2.7 ⁇ m) using a linear gradient of (A) ACN + 0.1% formic acid to (B) water + 0.1% formic acid at a flow rate of 500 ⁇ l/min and a column temperature of 45°C.
  • the gradient conditions are as follows: 0-0.5 min, 50% B; 0.5-2 min, 50-37% B; 2-4 min, 37-50% B, 4- INM-426 INMPT22046DEWO 11/07/2024 5 min 50% B at 1500 ⁇ l/min (column cleaning), 5-7 min 50% B down to 500 ⁇ l/min.
  • the LC stream is fed into the dual AJS-ESI source, which is set to a capillary voltage of 4000 V, a nebulizer gas pressure of 50 psi, a dry gas flow of 12 l/min, and a dry gas temperature of 350 °C.
  • the TOF parameters used are extended dynamic matic range (2 GHz), 160 V fragmentor and 45 V skimmer voltage
  • the mass spectra were recorded at a time interval of 0.75-2 minutes in full-scan mode in the m/z range 100-1000 with a spectral rate of 1/s.
  • the negatively charged mass [M-H]- at m/z 255.0667 Da was extracted and automatically integrated using the Mass Hunter software.
  • coli Nissle 1917 is a commercial probiotic (Mutaflor) that has been extensively researched as a chassis for drug delivery in the body (Chen et al., 2023).
  • Motaflor a commercial probiotic
  • pinocembrin production in cultures containing cinnamic acid was observed only with the strain containing the P K2 promoter.
  • the inserts could not be detected in the sequencing results when plasmids with the P OY and P R10 promoters in E. coli Nissle 1917, which indicates a possible incompatibility of these constructs with the strain indicates.
  • PVA is a synthetic, water-soluble, and biocompatible polymer widely used for the encapsulation of probiotics ( ⁇ anga, Dudak, 2021; Corona-Escalera et al., 2022).
  • the PVA backbone was modified with vinylsulfone groups. modified to form a stable hydrogel by chemical cross-linking. network.
  • Bacterial hydrogels that contain E. coli Nissle 1917, encapsulated in PVA-VS, were contained in perforated plates and photocrosslinked.
  • coumaric acid was converted into naringenin, caffeic acid into eriodictyol, ferulic acid into homoeriodictyol, and various cinnamic acid analogues into corresponding pinocembrin analogues.
  • the ELM concept of the composite material according to the invention could therefore potentially be used for the selective production of a range of bioactive compounds depending on the input starting materials.
  • a limitation of this example is the low efficiency the conversion of cinnamic acid to pinocembrin. With the current, non-optimized enzyme cascade, the amount of the product pinocembrins (100 - 400 ng/mL) by three orders of magnitude lower than that of the precursor (20 - 600 ⁇ g/mL).
  • ninaceous acid >95% purity
  • pinocembrin >95% purity
  • the conversion efficiency of the current system could offer a cost advantage if it can be maintained in the body.
  • studies have been carried out based on vitro cell culture and in vivo animal studies 10 to 100 times higher here concentrations reported (Soromou et al., 2013; Gong, INM-426 INMPT22046DEWO 11/07/2024 2021). Therefore, an improvement in conversion efficiency may still be required.
  • pinocembrin production in E. coli 18-fold to up to 40 ⁇ g/mL was reported by regulating cinnamic acid metabolism (Cao et al., 2016).
  • pinocembrin yield could be increased to almost 200 ⁇ g/mL by modifying the antibiotic resistance cassette to kanR from ampR. and a CoA ligase from soybeans was used (Dustan et Such optimization could improve the efficiency of precursor conversion and the production rate of the bioactive compound from the ELM. Nevertheless, the currently achieved low production yields could still offer the possibility of using these ELMs to achieve highly localized, microdosed effects in the body, such as anti-inflammatory and antioxidant effects.
  • the matrix according to the invention allows reliable control of the growth and encapsulation of microorganisms.
  • ELMS For application in the body, ELMS with formats compatible with the tissue type are required.
  • PVA hydrogels can be produced in various formats, e.g., as microcapsules (Han, Hong, 2006), electrospun meshes (Akbar et al., 2018), or 3D-printed scaffolds (Meng et al., 2020).
  • These diverse possibilities underscore the potential of catalytic ELMs to be developed as living medical devices that can promote health cost-effectively.
  • An example of a hyaluronic acid-producing construct is given in Table 6. This can, for example, be used in Coryne- bacterium glutamicum.
  • the construct allows the production and release of hyaluronic acid in C. glutamicum.
  • Rho kinase inhibition activity of pinocembrin in rat aortic rings contracted by angiotensin II Chin J Nat Med 2013, 11(3):258-63 Li, X.; Zhai, Y.; Xi, B.; Ma, W.; Zhang, J.; Ma, X.; Miao, Y.; Zhao, Y.; Ning, W.; Zhou, W.; Yang, C. Pinocembrin Ameliorates Skin Fibrosis via Inhibiting TGF- ⁇ 1 Signaling Pathway.
  • Pinocembrin protects SH-SY5Y cells against MPP+-induced neurotoxicity through the mitochondrial apoptotic pathway.
  • J Mol Neurosci 2014, 53(4):537-45 Wang, Y.; Liu, Y.; Li, J.; Chen, Y.; Liu, S.; Zhong, C. Engineered living materials (ELMs) design: From function assignment INM-426 INMPT22046DEWO 07.11.2024 on dynamic behavior modulation. Current Opinion in Chemical Biology 2022, 70 Wu, X.; Liu, J.; Liu, D.; Yuwen, M.; Koffas, MAG; Zha, J.
  • INM-426 INMPT22046DEWO 07.11.2024 Table 1: Physicochemical properties of PVA-VS/PVA hydrogels and comparison with properties of commercial PVA contact lenses, measured under the same conditions: Mate- Pol- Eq- Oxygen- Water- Trans- Refractive- Spei- rmo- u s G' a) 6 ⁇ 0.2 9 ⁇ 0.1 ⁇ 0.3 ⁇ 0.2 ⁇ 0.1 a) Calculated from EWC measurements; b) Value at 600 nm; c) Obtained from time sweep measurements.
  • the table also includes the molecular weight of the proteins used for the experiment and the band assignment in Figure 7 b) a) The band corresponding to aldolase was detected at 40 kDa (Mw of its subunits) due to denaturation of the protein by dodecyl sulfate.
  • INM-426 INMPT22046DEWO 07.11.2024 Table 3: Plasmids used in the examples: 5 INM-426 INMPT22046DEWO 07.11.2024 Table 4:
  • INM-426 INMPT22046DEWO 07.11.2024 Table 5 Synthetic DNA fragments for subcloning. Promoter regions are shown in capital letters, while terminators are underlined. INM-426 INMPT22046DEWO 07.11.2024 Table 6: Sequence of a hyaluronic acid-producing construct. Promoter Peftu (italics), hasA (codon-optimized, from Streptomyces equisimilis ), ribosomal binding site (bold), and hasB INM-426 INMPT22046DEWO 11/07/2024

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Botany (AREA)
  • Dermatology (AREA)
  • Transplantation (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Birds (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medical Informatics (AREA)
  • Molecular Biology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

L'invention concerne un matériau composite pour produits médicaux, comprenant a) au moins un matériau de matrice, et b) au moins un micro-organisme incorporé dans le matériau de matrice, le ou les micro-organismes pouvant libérer au moins une substance dans l'environnement du matériau composite.
PCT/EP2024/081545 2023-11-09 2024-11-07 Matériau composite vivant pour produits médicaux Pending WO2025099175A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE102023131080.1 2023-11-09
DE102023131080 2023-11-09
DE102023131356.8A DE102023131356A1 (de) 2023-11-09 2023-11-10 Lebendes Kompositmaterial für medizinische Produkte
DE102023131356.8 2023-11-10

Publications (1)

Publication Number Publication Date
WO2025099175A1 true WO2025099175A1 (fr) 2025-05-15

Family

ID=93563215

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/081545 Pending WO2025099175A1 (fr) 2023-11-09 2024-11-07 Matériau composite vivant pour produits médicaux

Country Status (1)

Country Link
WO (1) WO2025099175A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0935523B1 (fr) * 1996-10-28 2004-09-29 Inc. General Mills Inclusion et encapsulation de particules a liberation controlee
US9518188B2 (en) 2010-11-15 2016-12-13 John S. Lettow Method of printing a conductive article and articles made thereby

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0935523B1 (fr) * 1996-10-28 2004-09-29 Inc. General Mills Inclusion et encapsulation de particules a liberation controlee
US9518188B2 (en) 2010-11-15 2016-12-13 John S. Lettow Method of printing a conductive article and articles made thereby

Non-Patent Citations (49)

* Cited by examiner, † Cited by third party
Title
AKBAR, Z.ZAHOOR, T.HUMA, N.JAMIL, A.AYESHA, H.KUMAR IRUDAYARAJ, J. M: "Electrospun probiotics: an alternative for encapsulation", J BIOL REGUL HOMEOST AGENTS, vol. 32, no. 6, November 2018 (2018-11-01), pages 1551 - 1556
BHUSARI, S.KIM, J.POLIZZI, K.SANKARAN, S.DEL CAMPO, A: "Encapsulation of bacteria in bilayer Pluronic thin film hydrogels: A safe format for engineered living materials", BIOMATER ADV, vol. 145, February 2023 (2023-02-01), pages 213240
BHUSARI, S.SANKARAN, S.DEL CAMPO, A: "Regulating Bacterial Behavior within Hydrogels of Tunable Viscoelasticity", ADV SCI, vol. 9, no. 17, June 2022 (2022-06-01), pages e2106026
CANGA, E. M.DUDAK, F. C: "Improved digestive stability of probiotics encapsulated within poly(vinyl alcohol)/cellulose acetate hybrid fibers", CARBOHYDR POLYM, vol. 264, 15 July 2021 (2021-07-15), pages 117990
CAO, W. ET AL.: "Enhanced pinocembrin production in Escherichia coli by regulating cinnamic acid metabolism", SCI REP, vol. 6, 2 September 2016 (2016-09-02), pages 32640
CHEN, H. ET AL.: "Advances in Escherichia coli Nissle 1917 as a customizable drug delivery system for disease treatment and diagnosis strategies", MATER TODAY BIO, vol. 18, 6 January 2023 (2023-01-06), pages 100543
CONDI MAINARDI JESSICA ET AL: "Embedding live bacteria in porous hydrogel/ceramic nanocomposites for bioprocessing applications", BIOPROCESS AND BIOSYSTEMS ENGINEERING, SPRINGER, DE, vol. 42, no. 7, 5 April 2019 (2019-04-05), pages 1215 - 1224, XP036827381, ISSN: 1615-7591, [retrieved on 20190405], DOI: 10.1007/S00449-019-02119-4 *
CORONA-ESCALERA, A. F. ET AL.: "Enzymatisch vernetzte Hydrogele aus Gelatine und Poly(vinylalkohol), beladen mit probiotischen Bakterien als orales Verabreichungssystem", PHARMAZIE, vol. 14, no. 12, 9 December 2022 (2022-12-09)
DEMERLIS, C. C.SCHONEKER, D. R: "Review of the oral toxicity of polyvinyl alcohol (PVA", LEBENSMITTEL CHEM TOXICOL, vol. 41, no. 3, March 2003 (2003-03-01), pages 319 - 26, XP029990173, DOI: 10.1016/S0278-6915(02)00258-2
DHAKANE, P.TADIMARRI, V. S.SANKARAN, S: "Light-Regulated Pro-Angiogenic Engineered Living Materials", FORTGESCHRITTENE FUNKTIONELLE MATERIALIEN, vol. 33, no. 31, May 2023 (2023-05-01)
DUSTAN, M.S. ET AL.: "Engineering Escherichia coli towards de novo production of gatekeeper (2 S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol", SYNTH BIOL, 6 August 2020 (2020-08-06), pages 5
GONG, H: "Pinocembrin unterdrückt die Proliferation und steigert die Apoptose in Lungenkrebszellen in vitro durch Hemmung der Autophagie", BIOENGINEERED, vol. 12, no. 1, December 2021 (2021-12-01), pages 6035 - 6044
HAN CHUNLI ET AL: "Hydrogel microcapsules containing engineered bacteria for sustained production and release of protein drugs", BIOMATERIALS, vol. 287, 5 June 2022 (2022-06-05), AMSTERDAM, NL, XP093242406, ISSN: 0142-9612, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S0142961222002599> [retrieved on 20250123], DOI: 10.1016/j.biomaterials.2022.121619 *
HAN, Y. K.HONG, S. C: "Preparation and Application of Poly(Vinyl Alcohol) Microcapsules Containing Azo Dye", MOLEKULARE KRISTALLE UND FLÜSSIGKRISTALLE, vol. 349, no. 1, September 2006 (2006-09-01), pages 79 - 82
HEIM, K.E.TAGLIAFERRO, A.R.BOBILYA, D.J: "Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships", J NUTR BIOCHEM, vol. 13, no. 10, 2002, pages 572 - 584, XP055206323, DOI: 10.1016/S0955-2863(02)00208-5
HOEFGEN, S. ET AL.: "Facile assembly and fluorescence-based screening method for heterologous expression of biosynthetic pathways in fungi", METAB ENG, vol. 48, July 2018 (2018-07-01), pages 44 - 51, XP085977761, DOI: 10.1016/j.ymben.2018.05.014
HUANG, Y.HOEFGEN, S.GHERLONE, F.VALIANTE, V: "Intrinsic Ability of the β-Oxidation Pathway To Produce Bioactive Styrylpyrones", ANGEW CHEM INT ED ENGL, vol. 61, no. 34, 22 August 2022 (2022-08-22), pages e202206851
JIANG, S.LIU, S.FENG, W: "PVA hydrogel properties for biomedical application", J MECH BEHAV BIOMED MATER, vol. 4, no. 7, October 2011 (2011-10-01), pages 1228 - 33, XP028242702, DOI: 10.1016/j.jmbbm.2011.04.005
KUFS, J.E.; HOEFGEN, S.; RAUTSCHEK, J.; BISSELL, A.U.; GRAF,C.; FIEDLER, J.; BRAGA, D.; REGESTEIN, L.; ROSENBAUM, M.A.;THIELE, J.;: "Rational Design of Flavonoid Production Routes Using Combinatorial and Precursor-Directed Biosynthesis", ACS SYNTH. BIOL., vol. 9, no. 7, 2020, pages 1823 - 1832
LI LIANYUE ET AL: "Hydrogel-Encapsulated Engineered Microbial Consortium as a Photoautotrophic "Living Material" for Promoting Skin Wound Healing", APPLIED MATERIALS & INTERFACES, vol. 15, no. 5, 28 January 2023 (2023-01-28), US, pages 6536 - 6547, XP093242400, ISSN: 1944-8244, Retrieved from the Internet <URL:https://pubs.acs.org/doi/pdf/10.1021/acsami.2c20399> [retrieved on 20250123], DOI: 10.1021/acsami.2c20399 *
LI, L.YANG, H.YUAN, T.ZHAO, Y.DU, G: "Rho kinase inhibition activity of pinocembrin in rat aortic rings contracted by angiotensin II", CHIN J NAT MED, vol. 11, no. 3, 2013, pages 258 - 63
LI, X.ZHAI, Y.XI, B.MA, W.ZHANG, J.MA, X.MIAO, Y.ZHAO, Y.NING, W.ZHOU, W.: "Pinocembrin Ameliorates Skin Fibrosis via Inhibiting TGF-βl Signaling Pathway", BIOMOLECULES, vol. 11, no. 8, 2021, pages 1240
LIU, R.; LI, J.; SONG, J.; ZHOU, D.; HUANG, C.; BAI, X.; XIE,T.; ZHANG, X.; LI, Y.; WU, C.; ZHANG, L.; LI, L.; ZHANG, T.;DU, G.: "Pinocembrin verbessert die Kognition und schützt die neurovaskuläre Einheit bei Alzheimer-bezogenen Defiziten", NEUROBIOL AGING, vol. 35, no. 6, 2014, pages 1275 - 85
LU, Q.LI, L.XUE, S.YANG, D.WANG, S: "Stability of Flavonoid, Carotenoid, Soluble Sugar and Vitamin C in 'Cara Cara' Juice during Storage", FOODS, vol. 8, no. 9, 2019, pages 417
LYNCH, J. PGOERS, L.LESSER, C. F: "Emerging strategies for engineering Escherichia coli Nissle 1917-based therapeutics", TRENDS PHARMACOL SCI, vol. 43, no. 9, September 2022 (2022-09-01), pages 772 - 786, XP087146262, DOI: 10.1016/j.tips.2022.02.002
MA, T.ZHANG, H.LI, T.BAI, J.WU, Z.CAI, T.CHEN, Y.XIA, X.DU, Y.FU, W: "Protective effect of pinocembrin from Penthorum chinense Pursh on hepatic ischemia reperfusion injury via regulating HMGB1/TLR4 signal pathway", PHYTOTHER RES., vol. 37, no. 1, January 2023 (2023-01-01), pages 181 - 194
MENG, Y.CAO, J.CHEN, Y.YU, Y.YE, L: "3D printing of a poly(vinyl alcohol)-based nano-composite hydrogel as an artificial cartilage replacement and the improvement mechanism of printing accuracy", J MATER CHEM B., vol. 8, no. 4, 28 January 2020 (2020-01-28), pages 677 - 690
OHEMENG, K. A.SCHWENDER, C. F.FU, K. P.BARRET, J. F: "DNA gyrase inhibitory and antibacterial activity of some flavones(1", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 3, no. 2, 1993, pages 225 - 230, XP055073066, DOI: 10.1016/S0960-894X(01)80881-7
OKOYE, C. 0.JIANG, H.WU, Y.LI, X.GAO, L.WANG, Y.JIANG, J: "Bacterial biosynthesis of flavonoids: Überblick, aktuelle biotechnologische Anwendungen, Herausforderungen und Perspektiven", J CELL PHYSIOL, 6 April 2023 (2023-04-06)
PANDEY, R. P.PARAJULI, P.KOFFAS, M. A. G.SOHNG, J. K: "Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology", BIOTECHNOL ADV, vol. 34, no. 5, September 2016 (2016-09-01), pages 634 - 662, XP029637369, DOI: 10.1016/j.biotechadv.2016.02.012
PRAVESCHOTINUNT, P.; DURAJ-THATTE, A. M.; GELFAT, I.; BAHL, F.;CHOU, D. B.; JOSHI, N. S.: "Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut", NAT COMMUN, vol. 10, no. 1, 6 December 2019 (2019-12-06), pages 5580
PRIKS, H. ET AL.: "Physical Confinement Impacts Cellular Phenotypes within Living Materials", ACS APPL BIO MATER, vol. 3, no. 7, 20 July 2020 (2020-07-20), pages 4273 - 4281
RAYAMAJHI, V.DHAKAL, D.CHAUDHARY, A. K.SOHNG, J. K: "Improved production of 1-deoxynojirymicin in Escherichia coli through metabolic engineering", WORLD J MICROBIOL BIOTECHNOL, vol. 34, no. 6, 2018, pages 77
RIVERIA-TARAZONA, L.K.CAMPBELL, Z.T.WARE, T.H: "Stimuli-responsive engineered living materials", WEICHE MATERIE, vol. 17, no. 4, 28 January 2021 (2021-01-28), pages 785 - 809
RODRIGO-NAVARRO, A.SANKARAN, S.DEL CAMPO, A.: "Engineered living biomaterials", NATURE REVIEWS MATERIALS, vol. 6, 2021, pages 1175 - 1190, XP037634155, DOI: 10.1038/s41578-021-00350-8
RODRIGUES, A.L.TRACHTMANN, N.BECKER, J.LOHANATHA, A.F.BLOTENBERG, J.BOLTEN, C.J.KORNELI, C.DE SOUZA LIMA, A.O.PORTO, L.M.SPRENGER,: "Systems metabolic engineering of Escherichia coli for production of the antitumor drugs violacein and deoxyviolacein", METABOLIC ENGINEERING, vol. 20, 2013, pages 29 - 41, XP055823291, DOI: 10.1016/j.ymben.2013.08.004
SELEPE, M.A.VAN HEERDEN, F.A: "Application of the Suzuki-Miyaura reaction in the synthesis of flavonoids", MOLECULES, vol. 18, no. 4, 22 April 2013 (2013-04-22), pages 4739 - 65
SHENG, H.SUN, X.YAN, Y.YUAN, Q.WANG, J.SHEN, X: "Metabolic Engineering of Microorganisms for the Production of Flavonoids", FRONT BIOENG BIOTECHNOL, vol. 8, 7 October 2020 (2020-10-07), pages 589069
SHI, L.CHEN, B.GAO, M.ZHANG, H.LI, Y.WANG, L.DU, G: "The characteristics of therapeutic effect of pinocembrin in transient global brain ischemia/reperfusion rats", LIFE SCI, vol. 88, no. 11-12, 2011, pages 521 - 8
SOROMOU, L. W.; ZHANG, Y.; CUI, Y.; WEI, M.; CHEN, N.; YANG,X.; HUO, M.; BALDE, A.; GUAN, S.; DENG, X.; WANG, D.: "Subinhibitory concentrations of pinocembrin exert anti-Staphylococcus aureus activity by reducing α-toxin expression", J APPL MICROBIOL, vol. 115, no. 1, 2013, pages 41 - 9
TARTIK, M.LIU, J.TOUS MOHEDANO, M.MAO, J.CHEN, Y: "Optimizing yeast for high-level production of kaempferol and quercetin", MICROB CELL FACT, vol. 22, no. 1, 20 April 2023 (2023-04-20), pages 74
TOUS MOHEDANO, M.MAO, J.CHEN, Y: "Optimization of Pinocembrin Biosynthesis in Saccharomyces cerevisiae", ACS SYNTH BIOL, vol. 12, no. 1, 20 January 2023 (2023-01-20), pages 144 - 152
WANG, X.YANG, Y.AN, Y.FANG, G: "The mechanism of anticancer action and potential clinical use of kaempferol in the treatment of breast cancer", BIOMED PHARMACOTHER, vol. 117, 2019, pages 109086
WANG, Y.GAO, J.MIAO, Y.CUI, Q.ZHAO, W.ZHANG, J.WANG, H: "Pinocembrin schützt SH-SY5Y-Zellen gegen MPP+-induzierte Neurotoxizität durch den mitochondrialen apoptotischen Weg", J MOL NEUROSCI, vol. 53, no. 4, 2014, pages 537 - 45
WANG, Y.LIU, Y.LI, J.CHEN, Y.LIU, S.ZHONG, C: "Engineered living materials (ELMs) design: Von der Funktionszuweisung zur dynamischen Verhaltensmodulation", AKTUELLE MEINUNG IN CHEMISCHER BIOLOGIE, 2022, pages 70
WU, X.LIU, J.LIU, D.YUWEN, M.KOFFAS, M. A. G.ZHA, J: "Biosynthese von Eriodictyol aus Tyrosin durch Corynebacterium glutamicum", MICROB CELL FACT, vol. 21, no. 1, 14 May 2022 (2022-05-14), pages 86
YU SHUANGJIANG ET AL: "Hydrogels as promising platforms for engineered living bacteria-mediated therapeutic systems", MATERIALS TODAY BIO, vol. 16, 15 September 2022 (2022-09-15), XP093242411, ISSN: 2590-0064, Retrieved from the Internet <URL:https://www.sciencedirect.com/science/article/pii/S2590006422002332> [retrieved on 20250123], DOI: 10.1016/j.mtbio.2022.100435 *
ZHOU, J.DU, G.CHEN, J: "Novel fermentation processes for manufacturing plant natural products", CURR OPIN BIOTECHNOL, vol. 25, February 2014 (2014-02-01), pages 17 - 23
ZHOU, L.WANG, K.LI, L.LI, H.GENG, M: "Pinocembrin hemmt die Lipopolysaccharid-induzierte Produktion von Entzündungsmediatoren in BV2-Mikrogliazellen durch Unterdrückung des PI3K/Akt/NF-xB-Signalwegs", EUR J PHARMACOL, vol. 761, 2015, pages 211 - 6

Similar Documents

Publication Publication Date Title
Bayer Hyaluronic acid and controlled release: A review
Liu et al. Lignin‐based micro‐and nanomaterials and their composites in biomedical applications
CN108025110B (zh) 可注射大孔水凝胶
Liu et al. Alginate/gelatin‐based hydrogel with soy protein/peptide powder for 3D printing tissue‐engineering scaffolds to promote angiogenesis
US10995186B2 (en) Light-activated preparation of hydrogels
EP2029629B1 (fr) Hydrogels de mélanges de polysaccharides utilisés en génie tissulaire et en tant que vecteurs de composés actifs
Sapru et al. Non-immunogenic, porous and antibacterial chitosan and Antheraea mylitta silk sericin hydrogels as potential dermal substitute
Siritientong et al. Preliminary Characterization of Genipin‐Cross‐Linked Silk Sericin/Poly (vinyl alcohol) Films as Two‐Dimensional Wound Dressings for the Healing of Superficial Wounds
Nooeaid et al. Alginate/gelatine hydrogels: Characterisation and application of antioxidant release
Aboomeirah et al. Wet electrospun nanofibers-fortified gelatin/alginate-based nanocomposite as a single-dose biomimicking skin substitute
Punyamoonwongsa et al. Silk sericin semi‐interpenetrating network hydrogels based on PEG‐diacrylate for wound healing treatment
Lai Influence of solvent composition on the performance of carbodiimide cross-linked gelatin carriers for retinal sheet delivery
Hu et al. Construction of a composite hydrogel of silk sericin via horseradish peroxidase‐catalyzed graft polymerization of poly‐PEGDMA
US10576042B2 (en) Composition comprising polyelectrolyte complexes, methods and uses thereof
WO2025099175A1 (fr) Matériau composite vivant pour produits médicaux
Shajari et al. Thiolated gellan gum/polyethylene glycol diacrylate hydrogels containing timolol maleate-loaded chitosan nanoparticles for ophthalmic delivery
DE102023131356A1 (de) Lebendes Kompositmaterial für medizinische Produkte
Vuković et al. Development of κ‐Carrageenan/Gelatin pH‐Responsive Hydrogels for Potential Skin Regeneration Application
Gao et al. Fabrication of novel polysaccharides and glycerol monolaurate based camellia oil composite oleogel: Application in wound healing promotion
Zhang et al. Caudicles in vandoid orchids: a carotenoid-based soft material with unique properties
DE102023131358A1 (de) Selbst-schmierende Kontaktlinsen
Mao et al. Spirulina protein isolate/carboxymethyl chitosan based emulsion gel with pH/enzyme-responsive release of curcumin for enhanced infected wound healing
Laomeephol Phospholipid-and gold-induced gelation of Thai silk fibroin and its applications for cell and drug delivery
WO2025099176A1 (fr) Lentilles de contact autolubrifiantes
He et al. Chitosan-based glucose-responsive hydrogel with spatiotemporally controlled bactericidal release for diabetic wound regeneration via multimodal therapy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24808270

Country of ref document: EP

Kind code of ref document: A1