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WO2022029739A1 - Hydrogels de soie immobilisant l'anhydrase carbonique, leurs procédés de fabrication et leurs utilisations - Google Patents

Hydrogels de soie immobilisant l'anhydrase carbonique, leurs procédés de fabrication et leurs utilisations Download PDF

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WO2022029739A1
WO2022029739A1 PCT/IB2021/057328 IB2021057328W WO2022029739A1 WO 2022029739 A1 WO2022029739 A1 WO 2022029739A1 IB 2021057328 W IB2021057328 W IB 2021057328W WO 2022029739 A1 WO2022029739 A1 WO 2022029739A1
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silk fibroin
hydrogels
carbonic anhydrase
previous
hrp
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Inventor
Viviana PINTO RIBEIRO
Joaquim Miguel ANTUNES CORREIA OLIVEIRA
Joana Catarina DA SILVA CORREIA DE OLIVEIRA
Rui Luís GONÇALVES DOS REIS
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Association for the Advancement of Tissue Engineering and Cell Based Technologies and Therapies A4TEC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • 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
    • A61L27/3637—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 characterised by the origin of the biological material other than human or animal, e.g. plant extracts, algae
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01001—Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
    • C12Y111/01—Peroxidases (1.11.1)
    • C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase

Definitions

  • the present disclosure relates to silk fibroin hydrogels, preferably cross-linked by a dual-enzymatic reaction mediated by peroxidase and human carbonic anhydrases ( ⁇ -class), and methods of producing said composition.
  • the disclosed enzymatically cross-linked silk fibroin hydrogels immobilizing human carbonic anhydrases may be used in vivo and ex vivo, as artificial extracellular matrices, bioinks and scaffolds for tissue engineering applications, tissue disease three- dimensional (3D) in vitro models, biomedical and pharmacological applications, including drug release, drug discovery, drug screening, and cancer therapy.
  • tissue engineering applications tissue disease three- dimensional (3D) in vitro models
  • biomedical and pharmacological applications including drug release, drug discovery, drug screening, and cancer therapy.
  • Biomedical research has been evolving towards the design and fabrication of three-dimensional (3D) implantable systems as patient-specific approaches in the diagnosis, prognosis and treatment of diseases. Such technologies allow for drug discovery, drug screening, tissue engineering and regenerative medicine.
  • Enzyme- mediated in situ forming hydrogels are especially attractive for this subject, as they have the ability to be loaded with cells and/or bioactive agents, and present a high specificity and non-toxic nature capable of being implanted in vivo and without inducing adverse inflammatory responses.
  • Proteins and polymers containing tyrosine groups have been proposed for enzyme-mediated hydrogelation reactions promoted by the horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) complex.
  • HRP horseradish peroxidase
  • H2O2O2O2 hydrogen peroxide
  • silk fibroin protein has been used for developing HRP-cross-linked hydrogels for a plethora of tissue engineering and regenerative medicine applications, as 3D extracellular matrices (ECM) mimics for cancer research, or as 3D primitive microfluidic systems.
  • Silk fibroin is FDA approved and has gained a lot of attention in tissue engineering and regenerative medicine due to its excellent mechanical, biochemical, and cellular properties coupled with its biocompatibility, flexibility, degradation properties, water-based processing, and the presence of easily accessible chemical groups for functional modifications. Moreover, it contains ⁇ 5 mol% tyrosine groups favouring its use for the enzyme cross-linking reaction and in situ hydrogelation process. This novel class of enzymatically cross-linked silk fibroin hydrogels have shown timely/spatially-responsive properties and cell encapsulation ability.
  • the viscoelastic properties of hydrogels seem to benefit from the enzymatic cross-linking of silk fibroin, showing tuneable mechanical and injectable properties according to the protein/cross-linker concentrations.
  • the control of cell behaviour of the reported cell-laden hydrogels is far from desired. This is because the spontaneous random coil-to- ⁇ -sheet conformational transition of silk fibroin affects the structure and opacity of the hydrogels with repercussions on cell viability and apoptosis.
  • CA’s are responsible for generating an intraluminal pH gradient that goes from 8.2 to 6.2 in the spinning duct.
  • the CO2 pressure is simultaneously increased as the pH decrease and its accumulation may affect the non- repetitive C-terminal (CT) region on silk structure inducing fibroin aggregation and ⁇ - sheets formation.
  • CT C-terminal
  • Silk fibroin forms di-tyrosine cross-linking sites with CA providing stabilization effects on its activity. Simultaneously, the rigidity at each binding site between the two molecules increases affecting the entire protein structure and improving hydrogels stability.
  • Human CA’s ( ⁇ -class) have also been explored for its catalytic activity in normal and diseased tissues/cellular compartments with different pH’s and metabolic rates. They catalyse the reverse hydration of CO2 to produce HCO3- and H + , properties that drive many physiological processes of tissues to maintain their homeostasis.
  • ⁇ -CA’s have served as therapeutic tools or targets of several diseases, including epilepsy and obesity, pulmonary hypertension, cerebral ischemia, rheumatoid arthritis, and cancer.
  • CA was entrapped in a matrix of ultrasonically bonded hydroxyapatite microparticles which were coated with ⁇ -sheet structured silk fibroin.
  • the silk fibroin structure induced a protective effect over CA activity and conformational properties.
  • further studies should be conducted to reveal if such system may be applied for CO 2 sequestration in accelerating industrial enzymatic reactions.
  • the method of carbonic anhydrase immobilization inside the hydrogels described in this document is substantially different from the method described in the present disclosure, as the method disclosed in this document does not involve the use of carbonic anhydrases directly in aqueous silk fibroin solutions.
  • the alcohol/ethanol chemical treatment also hinders possible biomedical applications, considering the known deleterious effect of alcohol/ethanol on cells.
  • the desired applications of this devices are for environmental issues, for removing, converting, or fixing carbon dioxide in nature.
  • Document CN109824922 describes infrared-responsive hydrogel material and preparation method thereof. This document discloses a preparation method of hydrogel material including several steps.
  • SiO 2 -NH 2 -ICG (ICG, indocyanine green) is dispersed into a silk fibroin solution followed by the addition of HRP and H 2 O 2 to perform the enzymatic cross-linking reaction and obtain infra-red responsive hydrogels.
  • the method disclosed in this document relates to the infrared-responsive properties of the hydrogels prepared for photothermal conversion and applicable for the controlled release of medicines.
  • Documents PT106041 (A), PT107426 (B) describe silk fibroin-derived hydrogels methods and uses thereof.
  • Document WO18025186 describes the enzymatic cross-linking of silk fibroin using HRP and H2O2 to obtain a hydrogel as nerve guidance conduits.
  • this HRP- mediated silk fibroin hydrogel is only an intermediate step to fabricate tubular silk conduits with final crystalline structure.
  • the document describes a ⁇ -sheet conformation induction as a subsequent step to the hydrogel’s formation.
  • the application of the silk fibroin hydrogels is specifically disclosed for nerve guidance conduits products.
  • the documented silk solutions are of high concentrations (5 to 25 wt%).
  • Document WO2018225049 describes the peroxidase-mediated cross-linking method to form silk fibroin hydrogels used as inks in 3D printing.
  • the processing technologies and ionic incorporation are responsible for chemically inducing ⁇ -sheet conformation at the time of hydrogelation.
  • the application of the highly porous silk fibroin hydrogels is specifically disclosed for bone and osteochondral regeneration products.
  • the documented silk solutions are of high concentrations (10 to 20 wt%).
  • the disclosed carbonic anhydrases-immobilizing silk fibroin hydrogels may be used as artificial extracellular matrices, bioinks and scaffolds for tissue engineering applications, tissue disease three-dimensional (3D) in vitro models, biomedical and pharmacological applications, including drug release, drug discovery, drug screening, and cancer therapy.
  • the present disclosure relates to a new methodology that comprises a dual- enzymatic cross-linking strategy, mediated by peroxidase and carbonic anhydrases, preferably human carbonic anhydrases ( ⁇ -class), to form silk fibroin hydrogels of flexible microstructure and higher ⁇ -sheet content to be used as bioinks with implantable characteristics.
  • This methodology overcomes the problems previously pointed out for the enzymatically cross-linked silk fibroin hydrogels used as inks in 3D printing. [0019] Specifically, this methodology overcomes the issues previously described for the horseradish peroxidase (HRP)-cross-linked silk fibroin hydrogels formed in amorphous conformation and compromising the structural stability of 3D printed structures.
  • HRP horseradish peroxidase
  • Silk is a natural protein with excellent biocompatibility and biodegradability, extensively used as biomaterial for different tissue engineering and regenerative medicine applications.
  • the new formulation is based on the rapidly responsive horseradish peroxidase (HRP)-cross-linked silk fibroin hydrogels formed at physiological conditions and with potential use as implantable artificial biomimetic three-dimensional (3D) matrices.
  • HRP horseradish peroxidase
  • 3D three-dimensional
  • the method of producing carbonic anhydrases-immobilizing enzymatically-cross-linked silk fibroin hydrogel device comprises: i) The mixing of human carbonic anhydrases ( ⁇ -class) with initially produced aqueous silk fibroin solution, horseradish peroxidase solution (HRP type VI, 0.84 mg/mL) and peroxide solutions, i.e., hydrogen peroxide solution (H 2 O 2 , 0.36 wt.%) or calcium peroxide solution (CaO 2 , 12 mg/mL calcium content); ii) The hydrogelation at 37°C promoting the cross-linking reaction between the tyrosine groups of both silk fibroin and carbonic anhydrases; iii) Involvement in a CO 2 saturated environment in order to generate lower pH levels and induce the formation of ⁇ -sheet structures; iv) characterization of this process with respect
  • the present disclosure relates to a silk fibroin hydrogel composition comprising carbonic anhydrase, for use in tissue engineering, regenerative medicine or drug screening.
  • the hydrogel is enzymatically crosslinked by peroxidases, in particular horseradish peroxidase.
  • the hydrogel is enzymatically crosslinked by peroxidases in the presence of peroxide solutions, in particular hydrogen peroxide or calcium peroxide.
  • the concentration of silk fibroin ranges from 1-16 wt.%, preferably from 2-16 wt.%, more preferably 2-4 wt.%.
  • the carbonic anhydrase is an alpha-carbonic anhydrase, in particular a cytosolic alpha-carbonic anhydrase.
  • the carbonic anhydrase is immobilized into the silk fibroin hydrogel by di-tyrosine cross-linking between the enzyme’s tyrosine residues and the tyrosines present in the silk fibroin.
  • the ⁇ -sheet content of the silk fibroin hydrogels increases by the carbonic anhydrase immobilization and activation in a CO2-saturated environment.
  • the CO2-saturated environment is a CO2-saturated water or saturated CO2 atmosphere.
  • Another aspect of the present disclosure comprises the disclosed silk fibroin hydrogel composition for use in medicine.
  • the present disclosure also relates to a silk fibroin hydrogel composition as replacement biomaterials, drug delivery platform, cell delivery platform, platforms for 3D cell culture and disease modelling, cell culture platform, cell encapsulation, organ development and tissue growth, artificial extracellular matrices, bioinks, scaffolds, or other biomedical and biological applications.
  • Another aspect of the present disclosure relates to a method to prepare the disclosed silk fibroin hydrogel, the method comprising: obtaining a silk fibroin solution; adding a peroxidase, in particular horseradish peroxidase, and a peroxide solution, preferably hydrogen peroxide or calcium peroxide, to the silk fibroin solution; adding a carbonic anhydrase solution to the silk fibroin solution containing the peroxidase and the peroxide solution to obtain a pre-gel solution, wherein the carbonic anhydrase has a concentration of 1-900 ⁇ g/mL, preferably 5-900 ⁇ g/mL, more preferably 5-450 ⁇ g/mL; inducing hydrogelation by incubating the solution at 37°C for at least 30 minutes, preferably 30 minutes to 1 hour to obtain a hydrogel; incubating the obtained hydrogel in a CO2 saturated environment, preferably CO2-saturated water or saturated CO2 atmosphere, for at least 5 minutes, preferably for 5-20 minutes
  • the disclosed method further comprises a step of placing the pre-gel solution inside polydimethylsiloxane moulds, on tissue culture polystyrene coverslips or in 3D printing cartridges before inducing hydrogelation.
  • the horseradish peroxidase concentration ranges between 0.5-1 mg/ml, preferably is 0.84 mg/ml.
  • the peroxide solution is a hydrogen peroxide solution, preferably hydrogen peroxide solution with a concentration ranging from 0.2-0.4 wt.%, more preferably 0.36 wt.%.
  • the peroxide solution is calcium peroxide solution, with a calcium content ranging from 5-20 mg/mL, preferably with a calcium content of 12 mg/mL.
  • An aspect of the present disclosure relates to a composition comprising the disclosed silk fibroin hydrogel.
  • the present disclosure also relates to an artificial extracellular matrix, bioink, or scaffold for tissue engineering applications, comprising the disclosed silk fibroin hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS [0039]
  • the following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.
  • Figure 1 illustrates an embodiment of the process for producing the enzymatically cross-linked silk fibroin hydrogels immobilizing carbonic anhydrases.
  • A The hydrogelation reaction between HRP and H 2 O 2 /CaO 2 with the tyrosine groups of silk fibroin.
  • B The carbonic anhydrase (CA) immobilization into the hydrogels through forming di-tyrosine cross-linking sites with silk fibroin.
  • the silk fibroin conformational network from the (C) aqueous silk fibroin solution, to the (D) HRP-mediated cross- linking of silk fibroin with multi-points attachment between the immobilized carbonic anhydrase and the silk fibroin hydrogel matrix (pH 7.4 and 37°C), and the (E) higher ⁇ - sheet content on silk fibroin after CO2 exposure.
  • Figure 2 shows representative images of the enzymatically cross-linked silk fibroin hydrogels, with 4 wt.% concentration of silk fibroin (HRP-4SF), (A, B) with and (C, D) without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), and after immersion in a CO2-saturated water bath.
  • Hydrogels were prepared either with (A, C) H2O2 or (B, D) CaO2 as oxidizer.
  • Figure 3 demonstrates the (A-D) flexibility and the (E-H) rigidity of the HRP-4SF hydrogels, (A-D) with and (E-H) without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), and after immersion in CO2-saturated water.
  • Hydrogels were prepared either (A, B, E, F) with H2O2 or (C, D, G, H) CaO2 as oxidizer.
  • Figure 4 shows representative images of the enzymatically cross-linked silk fibroin hydrogels, with 12 wt.% concentration of silk fibroin (HRP-12SF), (A, B) with and (C, D) without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), and after immersion in a CO2-saturated water bath.
  • Hydrogels were prepared either (A, C) with H2O2 or (B, D) CaO2 as oxidizer.
  • Figure 5 shows the (A) 3D printing of carbonic anhydrase (CA-II, 50 ⁇ g/mL) immobilizing silk fibroin hydrogels prepared using 6 wt.% concentration of silk fibroin (HRP-6SF) (BioX 3D Bioprinter). 3D printed scaffolds obtained (B) before and (C) after immersion in CO 2 -saturated water for 4 hours (37°C). Hydrogels were prepared either with H 2 O 2 or CaO 2 as oxidizer.
  • CA-II carbonic anhydrase
  • Figure 6 shows an embodiment of attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) spectra for the HRP-4SF hydrogels, with (HRP-4SF/CA) and without (HRP-4SF) carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL).
  • a – HRP- 4SF hydrogels prepared with H 2 O 2 as oxidizer, after immersion into PBS (formulations HRP-4SF/CA_PBS and HRP-4SF_PBS) or CO 2 -saturated water (formulations HRP-4SF/CA_ CO 2 sat and HRP-4SF_ CO 2 sat).
  • D - HRP-4SF hydrogels prepared with CaO2 as oxidizer, after immersion into PBS (formulations HRP-4SF/CA_PBS and HRP-4SF_PBS) or CO2-saturated water (formulations HRP-4SF/CA_ CO2sat and HRP- 4SF_ CO2sat), 7 days after preparation.
  • ATR-FTIR spectra shows the amide I, amide II and amide III regions on silk fibroin.
  • Figure 7 shows an embodiment of ATR-FTIR spectra for the HRP-12SF hydrogels, with (HRP-12SF/CA) and without (HRP-12SF) carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL).
  • C - HRP-12SF hydrogels prepared with CaO2 as oxidizer after immersion into PBS (formulations HRP- 12SF/CA_PBS and HRP-12SF_PBS) or CO2-saturated water (formulations HRP-12SF/CA_ CO2sat and HRP-12SF_CO2sat).
  • D - HRP-12SF hydrogels prepared with CaO2 as oxidizer after immersion into PBS (formulations HRP-12SF/CA_PBS and HRP-12SF_PBS) or CO 2 - saturated water (formulations HRP-12SF/CA_ CO 2 sat and HRP-12SF_CO 2 sat), 7 days after preparation.
  • FIG. 8 shows an embodiment of intrinsic fluorescence excitation-emission spectra of HRP-6SF hydrogels, with (HRP-6SF/CA) and without (HRP-6SF) carbonic anhydrase immobilization (CA-II, 50 ⁇ g/mL), and after immersion for 1 hour in both (A, B) CO 2 -saturated water and (C, D) PBS solution. Spectra obtained for 6 wt.% concentration of silk fibroin solution and carbonic anhydrase solution (50 ⁇ g/mL) were used as control (A-D).
  • FIG. 9 shows an embodiment of intrinsic fluorescence excitation-emission spectra of HRP-6SF hydrogels, with (HRP-6SF/CA) and without (HRP-6SF) carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), and after immersion for 1 hour in both (A, B) CO2-saturated water and (C, D) PBS solution. Spectra obtained for 6 wt.% concentration of silk fibroin solution and carbonic anhydrase solution (450 ⁇ g/mL) were used as control (A-D).
  • Hydrogels were prepared either with H2O2 (A, C) or (B, D) CaO2 as oxidizer.
  • Figure 10 shows an embodiment of the carbonic anhydrase (CA-II: 50, 200, 450 ⁇ g/mL) (A) release and (B) immobilization efficiency into the enzymatically cross-linked silk fibroin hydrogels, immersed up to 48 hours in CO2-saturated water and PBS solution, at 37°C under agitation (60 rpm). Carbonic anhydrase-immobilizing hydrogels were prepared with 4 wt.% concentration of silk fibroin solution. Hydrogels were prepared either with H2O2 or CaO2 as oxidizer.
  • Figure 11 shows the drug release profile of Doxorubicin (DOX) from enzymatically cross-linked silk fibroin hydrogels prepared with 4 wt.% concentration of silk fibroin solution, with (HRP-4SF/CA) and without (HRP-4SF) carbonic anhydrase immobilization (CA-II, 50 ⁇ g/mL). Hydrogels were immersed up to 14 days in (A) CO2- saturated water and (B) PBS solution. Hydrogels were prepared either with H2O2 or CaO2 as oxidizer.
  • DOX Doxorubicin
  • Figure 12 shows an embodiment of the metabolic activity of U251 cells encapsulated in the enzymatically cross-linked silk fibroin hydrogels, with (CA-II: 50, 200, 450 ⁇ g/mL) and without (No CA-II) carbonic anhydrase immobilization, up to 7 days of culture.
  • Cell-laden hydrogels prepared with (A, B) 4 wt.% concentration of silk fibroin (HRP-4SF), and (C, D) 2 wt.% concentration of silk fibroin (HRP-2SF).
  • Hydrogels were prepared either with H 2 O 2 (A, C) or (B, D) CaO 2 as oxidizer.
  • Figure 13 shows the viability of U251 cells encapsulated in the HRP-4SF and HRP- 2SF hydrogels, with (CA-II: 50, 200, 450 ⁇ g/mL) and without (No CA-II) carbonic anhydrase immobilization, up to 7 days of culture. Live/dead assay was performed and observed under fluorescence microscopy (scale bar: 500 ⁇ m). Hydrogels were prepared either with H 2 O 2 or CaO 2 as oxidizer.
  • Figure 14 shows the metabolic activity normalized by DNA content of U251 cells encapsulated in the enzymatically cross-linked silk fibroin hydrogels, with (CA-II: 50 ⁇ g/mL) and without (No CA-II) carbonic anhydrase immobilization for 24 hours.
  • Cells cultured under normoxia and hypoxia conditions 50 ⁇ M of cobalt (II) chloride, CoCl2).
  • Cell-laden hydrogels prepared with 4 wt.% concentration of silk fibroin (HRP-4SF). Hydrogels were prepared either with H2O2 or CaO2 as oxidizer.
  • Figure 15 schematically exemplifies the extrusion process of silk from Bombyx mori silkworms, illustrating the pH values in the different parts of the gland influenced by the carbonic anhydrase’s activity.
  • Figure 16 schematically illustrates different embodiments of what the carbonic anhydrases-immobilizing enzymatically crosslinked silk fibroin hydrogels can be used for.
  • DETAILED DESCRIPTION [0056] The present disclosure relates to dual-enzymatic cross-linked silk fibroin hydrogels and methods of producing the said composition.
  • the present disclosure relates to a silk fibroin hydrogel comprising carbonic anhydrase for use in tissue engineering, regenerative medicine or drug screening.
  • Another aspect of the present disclosure relates to a cross-linked silk fibroin hydrogels immobilizing human carbonic anhydrases, preferably enzymatically cross- linked, that may be used as artificial extracellular matrices, bioinks and scaffolds for tissue engineering applications, tissue disease three-dimensional (3D) in vitro models, biomedical and pharmacological applications, including drug release, drug discovery, drug screening, and cancer therapy.
  • the method to prepare said silk fibroin hydrogel is also disclosed.
  • the silk fibroin is initially extracted from Bombyx mori cocoons, by removing the sericin in a boiling solution of 0.02 M Na 2 CO 3 for at least 1 hour, and then rinsed in distilled water.
  • aqueous silk fibroin solution is prepared with concentrations of 1-16 wt.%, preferably from 2-16 wt.%, more preferably 2-4 wt.%. After dialysis the yield concentration of silk fibroin is about 6-8 wt.%.
  • Silk fibroin may be concentrated in a 20 wt.% of polyethylene glycol (PEG) solution or diluted in distilled water or phosphate-buffered saline (PBS) buffer solution.
  • PEG polyethylene glycol
  • PBS phosphate-buffered saline
  • calcium peroxide solution preparation requires the use of 2 M HCl to drop the pH and allow the dissolution.
  • the calcium content in the solution can be determined using Inductively Coupled Plasma-Optical Emission Spectrometry.
  • the carbonic anhydrases-immobilizing silk fibroin hydrogels are produced through a dual-enzymatic cross-linking reaction mediated by horseradish peroxidase and the carbonic anhydrases ( ⁇ -class), using the following procedure: i) Add the horseradish peroxidase (HRP type VI, 0.84 mg/mL) and the peroxide solution, i.e.
  • the physicochemical characterization of the hydrogels may be determined, as well as cell behaviour, up to 7 days of culture.
  • Figure 1 illustrates the process for producing the enzymatically cross-linked silk fibroin hydrogels immobilizing carbonic anhydrases.
  • A The hydrogelation reaction between HRP and H2O2/CaO2 with the tyrosine groups of silk fibroin.
  • B The carbonic anhydrase immobilization into the hydrogels through forming di-tyrosine cross-linking sites with silk fibroin.
  • the silk fibroin conformational network from the (C) aqueous silk fibroin solution, to the (D) HRP-mediated cross-linking of silk fibroin with multi-points attachment between the immobilized carbonic anhydrase and the silk fibroin hydrogel matrix (pH 7.4 and 37°C), and the (E) higher ⁇ -sheet content on silk fibroin after CO2 exposure.
  • Figure 2 shows representative images of the enzymatically cross-linked silk fibroin hydrogels, with 4 wt.% concentration of silk fibroin (HRP-4SF), (A, B) with and (C, D) without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), and after immersion in a CO2-saturated water bath. It is possible to observe that using a 4 wt.% concentration of silk fibroin, the presence of carbonic anhydrase did not affect the opacity of the formed hydrogels.
  • the concentration and ⁇ -type of carbonic anhydrases may be adjusted for improving the hydrogels structural and mechanical properties.
  • the mechanical properties of the hydrogels may be tuned by adjusting the concentration of silk fibroin.
  • initially produced HRP-cross-linked silk fibroin hydrogels (HRP-SF) with amorphous conformation give way to silk fibroin hydrogels cross-linked by a dual-enzymatic reaction mediated by HRP and carbonic anhydrase (HRP-SF/CA), and capable of including higher ⁇ -sheet content.
  • Figure 3 demonstrates that after immersion in CO 2 -saturated water, the HRP-4SF hydrogels present, (A-D) flexible and glue-like properties with the carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), whereas the non-immobilized hydrogels are more (E- H) rigid and less mouldable.
  • the flexibility and rigidity of the enzymatically cross-linked silk fibroin hydrogels were assessed by naked eye, while the hydrogels were handled using a scalpel.
  • FIG 4 shows representative images of enzymatically cross-linked silk fibroin hydrogels, with 12 wt.% concentration of silk fibroin (HRP-12SF), (A, B) with and (C, D) without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), and after immersion in a CO2-saturated water bath. It is possible to observe that using a 12 wt.% concentration of silk fibroin, the presence of carbonic anhydrase has effects on hydrogels opacity. This is because at high concentrations the transparency of the amorphous and non- immobilized silk fibroin hydrogels is superior as compared to those of low concentrations (i.e. HRP-4SF hydrogels).
  • the opacity of silk fibroin structures is an indicator of a main ⁇ -sheet conformation state of the protein, showing that the carbonic anhydrase-immobilization and CO2 treatment induced conformational changes and high crystallinity to the hydrogels.
  • the HRP-SF hydrogels are more rigid, whereas the HRP-SF/CA hydrogels are more flexible and viscous (glue-like properties), which may be ideal for encapsulating cells and flowing through printing devices.
  • Figure 5 shows the (A) 3D printing of carbonic anhydrase (CA-II, 50 ⁇ g/mL) immobilizing silk fibroin hydrogels prepared using 6 wt.% concentration of silk fibroin (HRP-6SF). 3D printed scaffolds obtained (B) before and (C) after immersion in CO 2 - saturated water for 4 hours (37°C). [0073] In an embodiment, an increase in the carbonic anhydrase-immobilizing silk fibroin hydrogels opacity was observed after immersion in CO 2 -saturated water, confirming the indication that the CO 2 has effects on carbonic anhydrase activity inducing conformational changes and high crystallinity to the hydrogels.
  • CA-II carbonic anhydrase
  • the carbonic anhydrase-immobilizing hydrogels present adequate properties to be used as inks/bioinks for scaffolds production in tissue engineering and biomedical applications.
  • Figure 6 shows an embodiment of the chemical composition and structural conformation of the HRP-4SF hydrogels analysed by Fourier transform infrared spectroscopy (FTIR) under an attenuated total reflectance (ATR), with and without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL).
  • FTIR Fourier transform infrared spectroscopy
  • ATR attenuated total reflectance
  • control hydrogels prepared without carbonic anhydrase immobilization presented low ⁇ -sheet content and a main amorphous conformation after immersion in PBS and CO2-saturated water (visible from amide-I absorbance peak, and indicated in table 1) (A, C), which may indicate the potential of carbonic anhydrase as a ⁇ -sheet inducer upon the reaction with CO2.
  • Figure 7 shows an embodiment of ATR-FTIR spectra for the enzymatically cross- linked silk fibroin hydrogels, with 12 wt.% concentration of silk fibroin (HRP-12SF), with and without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL).
  • CA-II carbonic anhydrase immobilization
  • control hydrogels prepared without carbonic anhydrase immobilization presented low ⁇ -sheet content and a main amorphous conformation after immersion in PBS and CO 2 -saturated water (visible from amide-I and amide-II absorbance peaks, and indicated in table 2) (A, C), which may indicate the potential of carbonic anhydrase as a ⁇ -sheet inducer upon the reaction with CO 2 .
  • the immersion in CO2-saturated water for 20 minutes caused the pH reduction of the SF hydrogel solutions (tables 1 and 2), and its accumulation may have affected the silk fibroin structure inducing fibroin aggregation and ⁇ -sheets formation in the presence of carbonic anhydrase.
  • Table 1 pH measurements of the HRP-4SF hydrogels, with and without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), after immersion in CO 2 -saturated water for 20 minutes (PBS solution used as control).
  • Table 2 pH measurements of the HRP-4SF hydrogels, with and without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), after immersion in CO 2 -saturated water for 20 minutes (PBS solution used as control).
  • HRP-12SF hydrogels pH measurements of the HRP-12SF hydrogels, with and without carbonic anhydrase immobilization (CA-II, 450 ⁇ g/mL), after immersion in CO2-saturated water for 20 minutes (PBS solution used as control).
  • HRP-cross-linked silk fibroin hydrogels HRP-SF
  • HRP-SF/CA dual-enzymatic reaction mediated by HRP and carbonic anhydrase
  • the sensitivity of the hydrogels to the immobilized carbonic anhydrase is higher at high silk fibroin concentrations (at least up to 12 wt.%). It is possible to observe that the typical absorbance peaks amide-I, II and III shifts towards lower wavenumber which is characteristic of high ⁇ -sheet content.
  • the concentration and ⁇ -type carbonic anhydrases may be adjusted for tuning the ⁇ -sheet content at the hydrogels.
  • Figure 8 shows an embodiment of intrinsic fluorescence excitation-emission spectra of HRP-6SF hydrogels, with and without carbonic anhydrase immobilization (CA- II, 50 ⁇ g/mL), and after immersion for 1 hour in both (A, B) CO 2 -saturated water and (C, D) PBS solution. Spectra obtained for 6 wt.% concentration of silk fibroin solution and carbonic anhydrase solution (50 ⁇ g/mL) were used as control (A-D). Analysis shows that hydrogels immersed for 1 hour in both CO 2 -saturated water and PBS formed di-tyrosine cross-linking sites (visible from an increase of the peak emission intensity at ⁇ 405 nm).
  • the immobilization of carbonic anhydrase into silk fibroin hydrogels was confirmed by the higher intensity peaks at ⁇ 405 nm, as compared to silk fibroin hydrogels without carbonic anhydrase immobilization. These may indicate the additional formation of di-tyrosine cross-linking sites between CA-SF and multi-points attachment between the carbonic anhydrase and the silk fibroin hydrogel matrix.
  • the peaks emission typically detected on silk fibroin ( ⁇ 305 nm) and carbonic anhydrase ( ⁇ 310 nm) were observed on hydrogels formulations immersed in both CO2-saturated water and PBS.
  • Figure 9 shows an embodiment of intrinsic fluorescence excitation-emission spectra of HRP-6SF hydrogels, with and without carbonic anhydrase immobilization (CA- II, 450 ⁇ g/mL), and after immersion for 1 hour in both (A, B) CO2-saturated water and (C, D) PBS solution. Spectra obtained for 6 wt.% concentration of silk fibroin solution and carbonic anhydrase solution (450 ⁇ g/mL) were used as control (A-D). Analysis shows that hydrogels immersed for 1 hour in both CO2-saturated water and PBS formed di-tyrosine cross-linking sites (visible from an increase of the peak emission intensity at ⁇ 405 nm).
  • the lower intensity peaks detected at ⁇ 405 nm on the carbonic anhydrase-immobilizing hydrogels may indicate that at higher concentrations (as high as 450 ⁇ g/mL) saturated levels of enzyme may interfere with the cross-linking reaction.
  • this behavior was not observed for the HRP-6SF/CA hydrogels prepared with H2O2 as oxidizer and after immersion for 1 hour in CO2-saturated water.
  • the CO 2 effects on CA are potentiated which benefits the binding to SF in the cross-linking reaction, even at high concentrations (as high as 450 ⁇ g/mL).
  • the peaks emission typically detected on silk fibroin ( ⁇ 305 nm) and carbonic anhydrase ( ⁇ 310 nm) were observed on hydrogels formulations immersed in both CO 2 -saturated water and PBS.
  • Figure 10 shows an embodiment of the carbonic anhydrase (CA-II: 50, 200, 450 ⁇ g/mL) (A) release and (B) immobilization efficiency into the enzymatically cross-linked silk fibroin hydrogels, immersed up to 48 hours in CO 2 -saturated water and PBS solution, at 37°C under agitation (60 rpm). Carbonic anhydrase-immobilizing hydrogels were prepared with 4 wt.% concentration of silk fibroin solution. [0093] In an embodiment, the carbonic anhydrase-immobilizing hydrogels immersed in PBS solution presented a higher release of CA-II, when immobilized at higher concentrations (200, 450 ⁇ g/mL).
  • CA-II carbonic anhydrase
  • the immersion in CO2-saturated water may have induced an increase of ⁇ -sheet content and hydrogels crystallinity entrapping the carbonic anhydrase even when immobilized at saturated levels.
  • low amounts of carbonic anhydrase were released from the enzymatically cross-linked silk fibroin hydrogels immersed in both CO2-saturated water and PBS, confirmed by immobilization efficiency levels found to be almost 100% for the different CA-II concentrations.
  • Hydrogels were encapsulated with DOX at 250 ⁇ g/mL and immersed up to 14 days in (A) CO2-saturated water and (B) PBS solution.
  • the low and regular DOX release profiles observed over the 14 days of analysis for all hydrogels formulations indicate the enzymatically cross-linked silk fibroin hydrogels, with and without carbonic anhydrase immobilization, as being stable and suitable for controlled drug release.
  • the carbonic anhydrase-immobilizing hydrogels showed a tendency to release less DOX, as compared to the same hydrogel formulations without carbonic anhydrase immobilization.
  • carbonic anhydrase-immobilizing silk fibroin hydrogels showed great potential for drug screening and controlled drug release applications, in particular for cancer therapy, as exemplified by the sustained release of DOX.
  • Figure 12 shows the metabolic activity, measured by Alamar blue assay, of U251 cells encapsulated in the enzymatically cross-linked silk fibroin hydrogels, with (CA-II: 50, 200, 450 ⁇ g/mL) and without (No CA-II) carbonic anhydrase immobilization, up to 7 days of culture.
  • Cell-laden hydrogels prepared with (A, B) 4 wt.% concentration of silk fibroin (HRP-4SF), and (C, D) 2 wt.% concentration of silk fibroin (HRP-2SF).
  • U251 glioma cell- line derives from a human malignant glioblastoma multiforme (GBM) and is widely used as a GBM model.
  • GBM human malignant glioblastoma multiforme
  • cell-laden hydrogels were obtained by preparing a mixture of SF solution (2 wt.% or 4 wt.%), HRP solution (HRP type VI, 0.84 mg/mL), H2O2 solution (H2O2, 0.36 wt.%) or CaO2 solution (CaO2, 12 mg/mL calcium content), and CA solution (50, 200, 450 ⁇ g/mL), warmed in a water bath at 37°C.
  • the warmed mixture solution was homogeneously mixed with a U251 cell pellet and transferred into TCPS coverslips to reach 100 ⁇ L hydrogels containing 1 x 10 4 cells per hydrogel.
  • CA- immobilizing hydrogels and control hydrogels were left to complete gelation at 37°C in the CO2 incubator, and after gel formation, culture medium was added to each sample. Samples were collected for analysis at day 1, 3 and 7.
  • the carbonic anhydrase-immobilizing silk fibroin hydrogels support cell encapsulation and an increasing cell metabolic activity for at least 7 days, preferably when using H 2 O 2 as oxidizer.
  • the carbonic anhydrase-immobilizing silk fibroin hydrogels are fabricated to support the growth of cells, including but not limited to eukaryotic cells (cancer cells, stromal cells, musculoskeletal system, immune system, and endothelial cells). Other cell types may also be added as required.
  • the ⁇ -carbonic anhydrase (CA-II) immobilized has no cytotoxic effects at desired concentrations.
  • the dual-enzymatic cross-linked silk fibroin hydrogels may further comprise carbonic anhydrases inhibitors as therapeutic agents, pharmacologically acceptable excipients, pharmaco-delivery agents, and mixtures thereof.
  • Figure 13 shows the viability of U251 cells encapsulated in the HRP-4SF and HRP-2SF hydrogels, with (CA-II: 50, 200, 450 ⁇ g/mL) and without (No CA-II) carbonic anhydrase immobilization, up to 7 days of culture. Live/dead assay was performed and observed under fluorescence microscopy (scale bar: 500 ⁇ m).
  • the carbonic anhydrase-immobilizing silk fibroin hydrogels support the natural cancer cells agglomeration/clustering after at least 7 days.
  • the carbonic anhydrase-immobilizing hydrogels prepared with lower concentration of silk fibroin allow higher cancer cells clustering, which may indicate that the lower the concentration of silk protein, the more apt become the matrices for a free cellular behaviour.
  • the carbonic anhydrase-immobilizing silk fibroin hydrogel functions to mimic the extracellular matrix (ECM) of human body tissues. For example, it may serve as a physical support for incorporating isolated cells during in vitro culture and subsequent implantation. As the cell populations are able to grown and function normally in hydrogels with stable structural conformation, the cells produce their natural ECM for desired time-periods, and the hydrogels may then biodegrade.
  • ECM extracellular matrix
  • the biodegradation of the hydrogel device may be controlled by various manufacturing techniques known in the art.
  • U251 cells encapsulation in the carbonic anhydrase-immobilizing silk fibroin hydrogels may be promoted under hypoxia conditions that better simulate the tumour microenvironment and CO 2 exposure. In such conditions, the effect of carbonic anhydrase over cancer cells natural behaviour can be higher due to the hypoxic culture conditions.
  • Figure 14 shows an embodiment of the metabolic activity, measured by Alamar blue assay, normalized by DNA content of U251 cells encapsulated in the enzymatically cross-linked silk fibroin hydrogels, with CA- II (50 ⁇ g/mL, formulations HRP-4SF/CA_H 2 O 2 , and HRP-4SF/CA_CaO 2 ) and without carbonic anhydrase immobilization (formulations HRP-4SF_H 2 O 2 , and HRP-4SF_CaO 2 ) for 24 hours.
  • Cells were cultured under normoxia and hypoxia conditions, and cell-laden hydrogels were prepared with 4 wt.% concentration of silk fibroin (HRP-4SF).
  • hypoxia conditions were achieved by cobalt chloride-induced chemical hypoxia, using 50 ⁇ M of cobalt (II) chloride (CoCl2).
  • the presence of CoCl2 stabilizes hypoxia inducible factors 1 ⁇ and 2 ⁇ under normoxia conditions, thus mimicking the conditions of a hypoxic chamber.
  • hypoxia conditions triggered CA-II activity on the carbonic- anhydrase-immobilizing silk fibroin hydrogels prepared using H2O2 as oxidizer, as showed by a superior metabolic activity of U251 cells encapsulated in the HRP-4SF/CA hydrogels as compared to the HRP-4SF hydrogels.
  • carbonic-anhydrase-immobilizing silk fibroin hydrogels prepared using CaO2 as oxidizer, which can be related to the stronger cross-linking promoted by CaO2 requiring more time or higher CoCl2 concentrations for mimicking hypoxia conditions in these hydrogels.
  • carbonic anhydrase-immobilizing silk hydrogels are suitable as 3D in vitro models of glioblastoma, confirming that these matrices are capable of mimicking tissues’ ECM in which they naturally exhibit catalytic activity.
  • optimized 3D bioprinting of stable hydrogel matrices as: (i) Hierarchical scaffolds for regenerating multi-complex tissues of musculoskeletal system; (ii) 3D in vitro models mimicking the microenvironment of hypoxic tumours by encapsulating cancer cells, stem-like cancer cells, cancer stromal cells and microvascular endothelial cells inside the hydrogel matrices.
  • the term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

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Abstract

La présente invention concerne une composition d'hydrogel de fibroïne de soie comprenant de l'anhydrase carbonique destinée à être utilisée dans l'ingénierie tissulaire, la médecine régénérative ou le criblage de médicaments. La composition peut être utilisée in vivo et ex vivo, comme matrices extracellulaires artificielles, bio-encres et échafaudages pour des applications d'ingénierie tissulaire, modèles tridimensionnels (3D) in vitro de maladies tissulaires, applications biomédicales et pharmacologiques, y compris la libération de médicaments, la découverte de médicaments, le criblage de médicaments et la thérapie anticancéreuse. L'invention concerne également le procédé de préparation dudit hydrogel de fibroïne de soie.
PCT/IB2021/057328 2020-08-07 2021-08-09 Hydrogels de soie immobilisant l'anhydrase carbonique, leurs procédés de fabrication et leurs utilisations Ceased WO2022029739A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005012606A2 (fr) * 2003-04-10 2005-02-10 Tufts University Solution aqueuse concentree de fibroine et utilisation
KR20160116940A (ko) 2015-03-31 2016-10-10 포항공과대학교 산학협력단 탄산무수화효소가 고정된 실크 하이드로겔 및 이를 이용한 이산화탄소의 전환 또는 고정화
WO2018025186A1 (fr) 2016-08-01 2018-02-08 Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies A4Tec - Associação Conduits de guidage de nerfs, leurs procédés de production et leurs utilisations
KR101881587B1 (ko) * 2017-09-27 2018-07-24 주식회사 극동중앙연구소 3d 프린팅용 실크피브로인 구조체의 제조방법
WO2018215997A1 (fr) 2017-05-26 2018-11-29 Association For The Advancement Of Tissue Engineering Cell Based Technologies & Therapies (A4Tec) - Associação Procédés de production de composition dopée ionique et utilisations associées
WO2018225049A1 (fr) 2017-06-09 2018-12-13 Association For The Advancement Of Tissue Engineering Cell Based Technologies & Therapies (A4Tec) - Associação Encres pour impression 3d, leurs procédés de production et leurs utilisations
CN109824922A (zh) 2019-01-17 2019-05-31 湖北工程学院 一种具有红外光响应的水凝胶材料及其制备方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005012606A2 (fr) * 2003-04-10 2005-02-10 Tufts University Solution aqueuse concentree de fibroine et utilisation
KR20160116940A (ko) 2015-03-31 2016-10-10 포항공과대학교 산학협력단 탄산무수화효소가 고정된 실크 하이드로겔 및 이를 이용한 이산화탄소의 전환 또는 고정화
WO2018025186A1 (fr) 2016-08-01 2018-02-08 Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies A4Tec - Associação Conduits de guidage de nerfs, leurs procédés de production et leurs utilisations
WO2018215997A1 (fr) 2017-05-26 2018-11-29 Association For The Advancement Of Tissue Engineering Cell Based Technologies & Therapies (A4Tec) - Associação Procédés de production de composition dopée ionique et utilisations associées
WO2018225049A1 (fr) 2017-06-09 2018-12-13 Association For The Advancement Of Tissue Engineering Cell Based Technologies & Therapies (A4Tec) - Associação Encres pour impression 3d, leurs procédés de production et leurs utilisations
KR101881587B1 (ko) * 2017-09-27 2018-07-24 주식회사 극동중앙연구소 3d 프린팅용 실크피브로인 구조체의 제조방법
CN109824922A (zh) 2019-01-17 2019-05-31 湖北工程学院 一种具有红外光响应的水凝胶材料及其制备方法

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BENJAMIN P. PARTLOW ET AL: "Highly Tunable Elastomeric Silk Biomaterials", ADVANCED FUNCTIONAL MATERIALS, vol. 24, no. 29, 1 August 2014 (2014-08-01), DE, pages 4615 - 4624, XP055420680, ISSN: 1616-301X, DOI: 10.1002/adfm.201400526 *
COSTA JOÃO B. ET AL: "Fast Setting Silk Fibroin Bioink for Bioprinting of Patient-Specific Memory-Shape Implants", ADVANCED HEALTHCARE MATERIALS, vol. 6, no. 22, 1 November 2017 (2017-11-01), DE, pages 1701021, XP055866340, ISSN: 2192-2640, DOI: 10.1002/adhm.201701021 *
COSTA, J.B. ET AL.: "Fast setting silk fibroin bioink for bioprinting of patient-specific memory-shape implants", ADVANCED HEALTHCARE MATERIALS, vol. 6, no. 22, 2017, pages 1701021
DOMIGAN, L. ET AL.: "Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands", INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 65, 2015, pages 100 - 106
HAN, Y. ET AL.: "Silk fibroin-based hydrogels as a protective matrix for stabilization of enzymes against pH denaturation", MOLECULAR CATALYSIS, vol. 457, 2018, pages 24 - 32
HASTURK ONUR ET AL: "Enzymatically crosslinked silk and silk-gelatin hydrogels with tunable gelation kinetics, mechanical properties and bioactivity for cell culture and encapsulation", BIOMATERIALS, ELSEVIER, AMSTERDAM, NL, vol. 232, 23 December 2019 (2019-12-23), XP086001942, ISSN: 0142-9612, [retrieved on 20191223], DOI: 10.1016/J.BIOMATERIALS.2019.119720 *
KIM, C.S. ET AL.: "A bioinspired dual-crosslinked tough silk protein hydrogel as a protective biocatalytic matrix for carbon sequestration", NPG ASIA MATERIALS, vol. 9, no. 6, 2017, pages e391 - e391
LOPES J. H. ET AL: "Silk fibroin hydroxyapatite composite thermal stabilisation of carbonic anhydrase", JOURNAL OF MATERIALS CHEMISTRY A, vol. 3, no. 38, 24 August 2015 (2015-08-24), GB, pages 19282 - 19287, XP055866342, ISSN: 2050-7488, DOI: 10.1039/C5TA05397H *
LOPES, J. ET AL.: "Silk fibroin hydroxyapatite composite thermal stabilisation of carbonic anhydrase", JOURNAL OF MATERIALS CHEMISTRY A, vol. 3, no. 38, 2015, pages 19282 - 19287
RIBEIRO, V.P. ET AL.: "Rapidly responsive silk fibroin hydrogels as an artificial matrix for the programmed tumor cells death", PLOS ONE, vol. 13, no. 4, 2018, pages e0194441
YAN, L.-P. ET AL.: "Tumor growth suppression induced by biomimetic silk fibroin hydrogels", SCIENTIFIC REPORTS, vol. 6, 2016, pages 31037

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