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WO2018187831A1 - Substrats libérant de l'oxygène et compositions et utilisations associées - Google Patents

Substrats libérant de l'oxygène et compositions et utilisations associées Download PDF

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Publication number
WO2018187831A1
WO2018187831A1 PCT/AU2018/000054 AU2018000054W WO2018187831A1 WO 2018187831 A1 WO2018187831 A1 WO 2018187831A1 AU 2018000054 W AU2018000054 W AU 2018000054W WO 2018187831 A1 WO2018187831 A1 WO 2018187831A1
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Prior art keywords
oxygen
water
substrate
peroxide
substrate according
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Ceased
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PCT/AU2018/000054
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English (en)
Inventor
Anthony Richard BLENCOWE
Aurelien Forget
Frances HARDING
Neethu NINAN
Nicolas Hans VOELCKER
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University of South Australia
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University of South Australia
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Priority claimed from AU2017901352A external-priority patent/AU2017901352A0/en
Application filed by University of South Australia filed Critical University of South Australia
Publication of WO2018187831A1 publication Critical patent/WO2018187831A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B25/00—Layered products comprising a layer of natural or synthetic rubber
    • B32B25/04—Layered products comprising a layer of natural or synthetic rubber comprising rubber as the main or only constituent of a layer, which is next to another layer of the same or of a different material
    • B32B25/045—Layered products comprising a layer of natural or synthetic rubber comprising rubber as the main or only constituent of a layer, which is next to another layer of the same or of a different material of foam
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B25/00—Layered products comprising a layer of natural or synthetic rubber
    • B32B25/20—Layered products comprising a layer of natural or synthetic rubber comprising silicone rubber
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B5/00—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts
    • B32B5/18—Layered products characterised by the non- homogeneity or physical structure, i.e. comprising a fibrous, filamentary, particulate or foam layer; Layered products characterised by having a layer differing constitutionally or physically in different parts characterised by features of a layer of foamed material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2255/00—Coating on the layer surface
    • B32B2255/10—Coating on the layer surface on synthetic resin layer or on natural or synthetic rubber layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2255/00—Coating on the layer surface
    • B32B2255/26—Polymeric coating
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2255/00—Coating on the layer surface
    • B32B2255/28—Multiple coating on one surface
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2266/00—Composition of foam
    • B32B2266/02—Organic
    • B32B2266/0214—Materials belonging to B32B27/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2266/00—Composition of foam
    • B32B2266/06—Open cell foam
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2307/00—Properties of the layers or laminate
    • B32B2307/70—Other properties
    • B32B2307/724—Permeability to gases, adsorption
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2307/00—Properties of the layers or laminate
    • B32B2307/70—Other properties
    • B32B2307/726—Permeability to liquids, absorption
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2307/00—Properties of the layers or laminate
    • B32B2307/70—Other properties
    • B32B2307/726—Permeability to liquids, absorption
    • B32B2307/7265—Non-permeable
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2307/00—Properties of the layers or laminate
    • B32B2307/70—Other properties
    • B32B2307/73—Hydrophobic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2307/00—Properties of the layers or laminate
    • B32B2307/70—Other properties
    • B32B2307/732—Dimensional properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B2553/00—Packaging equipment or accessories not otherwise provided for

Definitions

  • the present disclosure relates to substrates and compositions that are capable of releasing oxygen upon contact with water, and uses thereof.
  • the present disclosure relates to biocompatible oxygen releasing devices for use in supplying oxygen to cells, tissues and/or organs in order to reduce the risk of hypoxia during isolation, transport and/or transplant processes.
  • Oxygen (dioxygen, 0 2 ) is vital for the existence of all multicellular organisms. Hypoxia occurs when such an organism lacks an adequate oxygen supply at the tissue level. Hypoxia can be a problem when cells, tissues or organs are removed from their natural environment. For example, organ transplantation often requires the transport of the graft tissue from an isolation center to a transplantation center where the recipient is located. As soon as the graft is isolated from the blood supply, a race against the clock commences. 1 The cells making up the graft tissue are deprived of oxygen and enter a hypoxic state. 2 This triggers biological pathways that eventually lead to cell death.
  • Biocompatible perfluorocarbon emulsions such as those containing perfluorooctyl bromide, have been added to the culture and shipping media to increase oxygen supply to cells. 9 11 These supplements increase the oxygen capacity of the media through complexation of oxygen with the perfluorocarbon. This process enhances oxygen delivery to the tissue and has been shown to improve the viability of grafts after transport. 12 However, perfluorocarbons can persist within the tissue, which can induce immune responses and other side effects. 13 ' 14 Alternatively, chemical reagents that generate oxygen upon reaction with water, such as calcium peroxide (Ca0 2 ) have been utilized to deliver oxygen to cells dispersed within a hydrogel matrix.
  • Ca0 2 calcium peroxide
  • the present disclosure results from research into the preparation of oxygen-releasing coatings by inserting either calcium peroxide or urea peroxide between layers of octadiene plasma polymer films.
  • the resultant substrates were found to release oxygen when contacted with water.
  • the oxygen release was measured and the substrates were found to improve cell survival under hypoxic conditions while generating limited amounts of toxic reactive oxygen species.
  • a substrate for sustaining isolated cells, tissues and/or organs by supplying oxygen thereto comprising at least one exposed surface comprising an oxygen generating material capable of chemically generating oxygen upon contact with water and a water and oxygen permeable membrane substantially covering the or each exposed surface.
  • a substrate for lowering the risk of hypoxia in isolated cells, tissues and/or organs comprising at least one exposed surface comprising an oxygen generating material capable of chemically generating oxygen upon contact with water and a water and oxygen permeable membrane substantially covering the or each exposed surface.
  • an oxygen generating substrate capable of releasing oxygen upon contact with water, the substrate comprising at least one exposed surface comprising an oxygen generating material capable of chemically generating oxygen upon contact with water and a water and oxygen permeable membrane substantially covering the or each exposed surface.
  • packaging suitable for transporting isolated cells, tissues and/or organs, the packaging including an oxygen generating substrate capable of releasing oxygen upon contact with water, the substrate comprising at least one exposed surface comprising an oxygen generating material capable of chemically generating oxygen upon contact with water and a water and oxygen permeable membrane substantially covering the or each exposed surface.
  • composition comprising an oxygen generating material and a water and oxygen permeable membrane wherein in the presence of water, the oxygen generating material reacts to release oxygen gas through the permeable membrane into a surrounding environment.
  • the substrate or composition of the first to fifth aspects comes into contact with water, diffusion of water through the membrane results in water contacting the oxygen generating material.
  • the oxygen generating material reacts with water to liberate oxygen which, in turn, is able to diffuse through the membrane after which it is released from the substrate or composition.
  • the substrate or composition is contained in a fixed volume the partial pressure of oxygen in the fixed volume then increases.
  • the substrate or composition is biocompatible.
  • Figure 1 shows different embodiments of a substrate of the present disclosure
  • Figure 2 shows schematically the fabrication of oxygen generating substrate; a base layer of plasma polymer film is initially deposited a PDMS disk, and then peroxide microparticles are deposited over the plasma polymer film. Finally, an outer plasma polymer coating is applied;
  • Figure 3 shows X-ray photoelectron spectra (A) for the unmodified PDMS surface and (B) the octadiene plasma polymer base layer. (C) Thickness of the deposited octadiene plasma polymer film for the base layer (1 deposition) and cumulative thickness of outer layers after 1, 2 and 3 deposition cycles;
  • Figure 5 shows (A) dissolved oxygen concentration over time produced in deoxygenated PBS at 20 °C by 21 ⁇ /mL of Ca0 2 and urea peroxide microparticles. (B) Dissolved oxygen concentration over time produced in deoxygenated PBS at 20 °C by different amounts of pure urea peroxide microparticles. Blank curve (red) show the background oxygen measured in the sealed container. (C) Dissolved oxygen concentration over time produced in deoxygenated PBS from oxygen generating substrates loaded with 2 mg (21 ⁇ ) of urea peroxide and coated with two or three outer plasma polymer layers;
  • Figure 6 shows fluorescence microscopy images of MIN6 cells stained with FDA for living cells (green) and with PI for dead cells (red) after 24 h of culture (A and B) under normoxia (21 % 02) and (C and D) hypoxia (0.5 % 02). Fluorescence microscopy images of MIN6 cells stained with FDA for living cells (green) and with PI for dead cells (red) after 24 h of culture under hypoxia (0.5 % 02) in the presence of urea peroxide oxygen generating substrates with (E and F) two and (G and H) three outer plasma polymer layers. Scale bars 200 ⁇ ; and
  • a substrate 10 that generates oxygen upon contact with an aqueous liquid.
  • the substrate 10 comprises an oxygen generating material 12 capable of chemically generating oxygen upon contact with water and a water and oxygen permeable membrane 14.
  • the water and oxygen permeable membrane 14 covers all or part of the surface of the oxygen generating material 12. If only part of the surface of the oxygen generating material 12 is covered by the water and oxygen permeable membrane 14 then the remainder of the surface is covered by a water impermeable material 16.
  • the substrate is said to comprise an "exposed surface” and the exposed surface 18 comprises the oxygen generating material.
  • the water and oxygen permeable membrane substantially covers the or each exposed surface.
  • an oxygen generating substrate 10 capable of releasing oxygen upon contact with water, the substrate 10 comprising at least one exposed surface comprising an oxygen generating material 12 capable of chemically generating oxygen upon contact with water and a water and oxygen permeable membrane 14 substantially covering the or each exposed surface.
  • a substrate 10 for sustaining isolated cells, tissues and/or organs by supplying oxygen thereto comprises at least one exposed surface comprising an oxygen generating material capable of chemically generating oxygen upon contact with water and a water and oxygen permeable membrane substantially covering the or each exposed surface.
  • the substrate for lowering the risk of hypoxia in isolated cells, tissues and/or organs.
  • the substrate comprises at least one exposed surface comprising an oxygen generating material 12 capable of chemically generating oxygen upon contact with water and a water and oxygen permeable membrane 14 substantially covering the or each exposed surface.
  • the packaging includes an oxygen generating substrate 10 capable of releasing oxygen upon contact with water.
  • the substrate 10 comprises at least one exposed surface comprising an oxygen generating material 12 capable of chemically generating oxygen upon contact with water and a water and oxygen permeable membrane 14 substantially covering the or each exposed surface.
  • the substrate 10 can take any form and may, for example, be in the form of a film, a disc, a capsule, a ball, etc.
  • the substrate may be part of a packaging material and may, for example, be coated onto a known thermoplastic material.
  • the substrate may be in the form of a disc, capsule or ball that may be included within packaging.
  • Substrate materials that can be used include polyolefins, such as polypropylene, polyethylene, polyisoprene, polybutadiene, and polybutene.
  • substrate materials that can be used include fluorinated polymers such as fluorinated ethylene propylene (FEP) polymers and polytetrafluorethylene (PTFE) polymers. Still further substrate materials that can be used include polysiloxanes, polycarbonates, polyamides, ethylene -vinyl acetate copolymers, ethylene- methacrylate copolymers, poly(vinyl chloride), polystyrene, polyesters, polyanhydrides, polyacrylianitrile, polysulfones, polyacrylic ester, acrylic, polyurethane and polyacetal, or copolymers or mixtures thereof. As discussed, the substrate may comprise a water impermeable material 16 which may be any one of the aforementioned thermoplastic materials, or another materials such as glass, metal, ceramic, etc.
  • FEP fluorinated ethylene propylene
  • PTFE polytetrafluorethylene
  • Still further substrate materials that can be used include polysiloxanes, polycarbonates, polyamides,
  • the oxygen generating material 12 can be any material that is capable of chemically generating oxygen upon contact with water. More specifically, the oxygen generating material 12 can be any substance capable of degrading to release hydrogen peroxide or any substance containing hydrogen peroxide. In certain embodiments, the oxygen generating material is a peroxide. Peroxides generate oxygen when they come into contact with water.
  • the peroxide is a metal peroxide, such as calcium peroxide (Ca0 2 ), lithium peroxide (Li 2 0 2 ), sodium peroxide (Na 2 0 2 ), beryllium peroxide (Be0 2 ), magnesium peroxide (Mg0 2 ), zinc peroxide (Zn0 2 ) or copper peroxide (Cu0 2 ).
  • the metal peroxide could also be any suitable peroxide of a group I or group II metal.
  • the metal peroxide may react with water to form hydrogen peroxide, which then degrades further to give 0 2 .
  • Other inorganic peroxides that could be used include percarbonates, such as sodium percarbonate.
  • the peroxide is a non-metallic peroxide complex, such as urea peroxide, ammonium peroxide or any other non-metallic complex like this has the potential to generate 0 2 .
  • the complexed hydrogen peroxide degrades to give 0 2 .
  • the oxygen generating material could be formed in situ by coating a substrate with a non-metallic compound capable of forming a complex with hydrogen peroxide and then contacting the coating with hydrogen peroxide solution to form a complex and then coating with the water and oxygen permeable membrane.
  • the oxygen generating material may be a solid, a liquid, a gel, an emulsion, etc
  • the oxygen generating material may be a neat material or it may be deposited on a support material, such as a sponge-like or open-celled foam of natural, synthetic, or mixed natural/synthetic origin.
  • the aqueous liquid can be any liquid that contains water.
  • the aqueous liquid can be water, saline solution, buffer solution, body fluids, water contained in tissues, etc.
  • the aqueous fluid may be naturally present in the organ or tissue and the water contained therein can react with the oxygen generating material to generate oxygen therefrom.
  • the water and oxygen permeable membrane is a polymer that allows for the controlled diffusion of water therethrough at a rate that allows oxygen to be generated at a desired rate.
  • the hydrophobic polymer may be a polyalkyl or a polyalkenyl homopolymer or copolymer.
  • the polymer is a plasma polymer.
  • Plasma polymerization is a simple, broadly applicable and environmentally friendly technique used for surface modification and coatings. 16 This coating technique allows for the deposition of a thin polymeric film (nanometer scale) to produce surfaces of different hydrophilicity and chemical functionality. 17 ' 18 Recently, we have demonstrated that levofloxacin can be sandwiched between two layers of plasma polymer films. Upon immersion of these films in cell culture medium, the drug was released as water diffused through the polymer films. 19 ' 2 "
  • the water and oxygen permeable membrane is a plasma obtained from any suitable organic compound such as alkane, alkene, alkyne or aromatic that can be plasma polymerised to form a hydrophobic material.
  • suitable organic compounds may include heteroatoms.
  • One suitable monomer for this purpose is 1,7-octadiene.
  • the water and oxygen permeable membrane is plasma polymerized 1,7 octadiene.
  • the octadiene plasma polymer is hydrophobic and thus diffusion of water through the film is retarded and controlled by the thickness of the film.
  • the release of oxygen from the substrate can be controlled by the plasma polymer deposition parameters. 18
  • the thickness of the outer plasma polymer layer it is possible to control water ingress and the oxygen release rate.
  • the thickness of the outer plasma polymer layer can be increased by successively depositing polymer films using the same plasma reactor parameters, thus providing a similar thickness for each deposition.
  • the thickness of the water and oxygen permeable membrane may be from about lOnm to about 150nm, such as about 40 nm, about 80 nm or about 120 nm.
  • the octadiene plasma polymer is biocompatible. Therefore, in embodiments the substrate or composition is biocompatible.
  • the oxygen generating substrate comprises oxygen generating material sandwiched between two water and oxygen permeable membrane layers.
  • the thickness of the oxygen generating substrate is between about 100 nm and about 100 ⁇ , such as between about 200 nm and about 40 ⁇ .
  • the oxygen generating substrates release sufficient oxygen to restore cell viability of hypoxic MIN6 cells. Results indicate that the oxygen generating substrate with two outer plasma polymer layers is optimal and can significantly improve cell survival under hypoxic conditions.
  • the oxygen generating devices and substrates described herein generate sufficient oxygen to support cell survival under hypoxia, such as about 0.5 % oxygen.
  • the oxygen generating substrates require the delivery of oxygen only and not the toxic hydrogen peroxide intermediate which can cause direct damage to cells and tissues, or further react to generate other reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • the thickness of the water and oxygen permeable membrane can be used to suppress the release of ROS from the oxygen generating device.
  • the oxygen generating devices and substrates described herein generate between 0% and 10% ROS.
  • Urea peroxide loaded oxygen generating substrates displayed reduced cell toxicity with a 50 % reduction in viable cell number for a single outer plasma polymer layer ( ⁇ 40 nm) and only a 20 % reduction in cell viability with two outer layers ( ⁇ 90 nm).
  • the experiments demonstrate that the water and oxygen permeable membrane layer needs to be thick enough to retain the hydrogen peroxide within the oxygen generating substrate for sufficient time for decomposition to occur.
  • composition comprising an oxygen generating material and a water and oxygen permeable membrane wherein in the presence of water, the oxygen generating material reacts to release oxygen gas through the permeable membrane into a surrounding environment.
  • PDMS disks were prepared from a 1 : 10 w/w precursor mixture of Sylgard® 184 Silicon
  • Plasma polymer deposition was performed in a custom built plasma reactor, described previously 4
  • the PDMS disks were placed into the plasma reactor, comprising a cylindrical stainless steel vacuum vessel with a diameter of 30 cm and a volume of ⁇ 20 L 4
  • the reactor was pumped down to a base pressure of 1 ⁇ 10 4 mbar using a two-stage rotary vane pump with a liquid N 2 cold trap.
  • oxygen was introduced into the chamber until a steady base pressure was achieved.
  • the plasma was ignited with a 50 W continuous wave (CW) radio frequency (RF) and left on for 20 min.
  • CW continuous wave
  • RF radio frequency
  • X-ray photoelectron spectroscopy (XPS).
  • a survey spectrum was recorded over the energy range 0 - 1000 eV using a pass energy of 100 eV and resolution of 0.5 eV using a take-off angle of 90° with respect to the sample surface.
  • a spot size of 3 mm was used.
  • Spectra analysis was performed using CasaXPS software.
  • PBS was deoxygenated by bubbling nitrogen through the solution until the concentration of dissolved oxygen was between 0 and 0.05 mg/L.
  • the oxygen generating substrate was then introduced into the PBS solution, the container was sealed and the oxygen concentration was measured over 400 min.
  • a control was performed in the sealed container by measuring the change of dissolved oxygen in the absence of the oxygen generating substrate.
  • MIN6 cell culture MIN6 cells were cultured in high-glucose Dulbecco ' s modified Eagle ' s medium (DMEM, Sigma) supplemented with 15 % v/v fetal bovine serum, 2.5 % v/v 1 M HEPES (Gibco), 1 % v/v glutamax (Gibco), 1 % v/v penicillin/streptomycin (Thermo Fisher), and 1 %v/v ⁇ - mercaptoethanol solution (5 ⁇ /L, Sigma). Once confluent, cells were trypsinized with a solution of 0.05 % trypsin EDTA (Sigma Aldrich).
  • Resazurin assay The biocompatibility of materials was assessed by quantifying the number of viable MIN6 cells using a resazurin assay.
  • MIN6 cells were seeded in 12 well plates at a density of 2 ⁇ 10 5 cells/mL in 2.5 mL of cell culture medium in each well. The cells were incubated for 24 h at 37 °C in contact with the PDMS disks with oxygen generating substrates, which floated on top of the cell medium with the thin film in contact with the cell medium. The cell culture medium was then replaced in each well with 250 of a stock solution of resazurin (1.5 mg/mL) and 2.5 mL of media.
  • DCFH-DA 2,7-dichlorodihydrofluorescein diacetate
  • hypoxia experiments Hypoxic conditions were created in a sealed plastic container with OxoidTM AnaeroGenTM (Thermo Fischer) anaerobic gas generating sachets.
  • MIN6 cells were seeded either under normoxia (21 % 0 2 ) or hypoxia (0.5 % 0 2 ) and incubated for 24 h at 37 °C in a 5 % C0 2 incubator. Subsequently, each sample was exposed to oxygen generating substrates of two and three layers thickness each loaded with 2 mg of urea peroxide for an additional 24 h, under hypoxic conditions. Finally, the cell viability was assessed with a resazurin assay as described previously, and FDA/PI live dead assay. Triplicates of each oxygen generating substrate were tested and data was plotted as ⁇ SD.
  • MIN6 cells were stained with fluorescein diacetate (FDA, Sigma Aldrich) and propidium iodide (PI, Sigma Aldrich).
  • FDA fluorescein diacetate
  • PI propidium iodide
  • MIN6 cells were seeded at a density of 2 x 10 5 cells/mL into a 12 well plate (400 ⁇ / ⁇ ).
  • PI and FDA solubilized in PBS was added to achieve a final concentration of 5 ⁇ g/mL and 5 ⁇ , respectively. Samples were incubated for 5-10 min prior to imaging. Fluorescence microscopy images were taken using a Nikon Eclipse TiS microscope.
  • Oxygen generating substrates were fabricated on PDMS substrates as they can be easily shaped to different sizes to allow incorporation within conventional labware. Hence, oxygen permeable PDMS disks (1.9 cm diameter) were molded to fit within 12 well culture plates. The PDMS disks were treated with an oxygen plasma to oxidize the surface and improve adhesion with the subsequently deposited octadiene plasma polymer base layer ( Figure 2). 21 Microparticles of urea peroxide or calcium peroxide (Ca0 2 ) were then manually deposited on the coated PDMS substrates.
  • the peroxides were mechanically milled and sieved to obtain particles with a size ⁇ 40 ⁇ .
  • An outer layer of octadiene plasma polymer was then deposited over the peroxide microparticles (Figure 3).
  • This " sandwich " coating was employed in order to ensure optimal adhesion of the plasma polymer coatings to the PDMS substrate, which overcomes instability of PDMS surface modification arising from chain mobility. 22 ' 23
  • the sandwich coating is independent of the substrate used and could be applied to any material compatible with the plasma polymer coating technique.
  • urea peroxide Two different oxygen-generating chemicals were used: the organic salt urea peroxide and the inorganic Ca0 .
  • Ca0 particles have previously been embedded within PDMS for the delivery of oxygen into cell cultures.
  • the main difference between urea peroxide and Ca0 is the reaction steps involved in the generation of oxygen upon contact with water (Table 1) and the formation of side -products. Ca0 initially reacts with water to generate hydrogen peroxide, which further decomposes to give water and oxygen. 24 During this process, Ca(OH) 2 is also formed, which even with its low solubility may be detrimental to cultured cells. In contrast, urea peroxide reacts after dissociation in one step with water to produce oxygen and non-toxic urea. 25
  • the thickness of the plasma polymer films was measured via ellipsometry of films deposited on silicon wafers, which revealed increasing thicknesses with successive deposition cycles (Figure 3C).
  • the first deposition cycle afforded a film thickness of 37 ⁇ 0.1 nm, with subsequent cycles providing similar increments to give total cumulative thicknesses of ⁇ 90 and 120 nm after 2 and 3 cycles, respectively.
  • Oxygen generating substrates loaded with Ca0 2 were found to induce complete cell death independent of the thickness of the plasma polymer coating.
  • the urea peroxide loaded oxygen generating substrates displayed reduced cell toxicity with a 50 % reduction in viable cell number for a single outer plasma polymer layer ( ⁇ 40 nm) and only a 20 % reduction in cell viability with two outer layers ( ⁇ 90 nm).
  • the experiments demonstrate that the outer plasma polymer layer needs to be thick enough to retain the hydrogen peroxide within the oxygen generating substrate for sufficient time for decomposition to occur.
  • the urea peroxide oxygen generating substrates with two or more outer plasma polymer layers were employed in subsequent experiments.
  • the number of viable MIN6 cells was quantified after exposure to the oxygen generating substrates and compared to the control (no oxygen generating substrate) under normoxic (21 % 0 2 ) and hypoxic (0.5 % 0 ) conditions using a resazurin assay (Figure 7).
  • the viability of the MIN6 cells exposed to the oxygen generating substrates displayed a statistically insignificant decrease compared to the control ( Figure 7A).
  • the slight level of cell death observed with the oxygen generating substrates under normoxic conditions may originate from an excess of oxygen within the cell culture media. Indeed, while under hypoxia an oxygen supplement is required, an excess of oxygen can be detrimental to cell health.
  • Oxygen generating substrates could be utilized to tackle the problem of hypoxia and hypoxia related apoptosis, especially for the transport of tissue grafts destined for transplantation.
  • DCFH-DA Dichloro-dihydro-fluorescein diacetate

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Abstract

L'invention concerne un substrat destiné à nourrir des cellules, des tissus et/ou des organes isolés. Le substrat nourrit des cellules, des tissus et/ou des organes isolés en leur fournissant de l'oxygène. Le substrat comprend au moins une surface apparente comprenant un matériau générant de l'oxygène pouvant générer chimiquement de l'oxygène au contact d'eau et une membrane perméable à l'eau et à l'oxygène recouvrant pratiquement la surface apparente ou chacune des surfaces apparentes.
PCT/AU2018/000054 2017-04-12 2018-04-12 Substrats libérant de l'oxygène et compositions et utilisations associées Ceased WO2018187831A1 (fr)

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WO2021260319A1 (fr) 2020-06-26 2021-12-30 Commissariat A L'energie Atomique Et Aux Energies Alternatives Dispositif passif de generation de dioxygene

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Publication number Priority date Publication date Assignee Title
CN109467155A (zh) * 2018-12-11 2019-03-15 同济大学 一种颗粒型过氧化钙缓释剂及其制备方法
WO2021260319A1 (fr) 2020-06-26 2021-12-30 Commissariat A L'energie Atomique Et Aux Energies Alternatives Dispositif passif de generation de dioxygene
FR3111883A1 (fr) 2020-06-26 2021-12-31 Commissariat A L'energie Atomique Et Aux Energies Alternatives Dispositif passif de generation de dioxygene

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