WO2018187831A1 - Oxygen releasing substrates and compositions and uses thereof - Google Patents

Abstract

A substrate for sustaining isolated cells, tissues and/or organs is disclosed. The substrate sustains isolated cells, tissues and/or organs by supplying oxygen thereto. The substrate 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.

Description

OXYGEN RELEASING SUBSTRATES AND COMPOSITIONS AND USES THEREOF

PRIORITY DOCUMENT

[0001] The present application claims priority from Australian Provisional Patent Application No. 2017901352 titled "OXYGEN RELEASING SUBSTRATES AND COMPOSITIONS AND USES THEREOF" and filed on 12 April 2017, the content of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to substrates and compositions that are capable of releasing oxygen upon contact with water, and uses thereof. In a particular form 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.

BACKGROUND

[0003] Oxygen (dioxygen, 0₂) 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.³ Therefore, the isolation, transport and transplantation processes face intense logistical pressures and must be precisely coordinated to avoid loss of graft viability prior to transplantation. The paucity of organs and tissues suitable for transplantation, and the high costs associated with the entire tissue transplant procedure, call for disruptive approaches that improve the quality and preservation time of isolated tissues.⁴'⁵

[0004] Currently, donor tissues are transported in sterile sealed bags in containers that maintain the graft at a constant sub-physiological temperature, typically between 4 and 20 °C.²'^6J At these temperatures, the metabolic rate and oxygen consumption of the graft tissue are reduced, lowering the risk of hypoxia and limiting cell death.⁸ While delivery of oxygen to the graft would further reduce the risk of hypoxia, it is difficult to implement this in a sterile autonomous container suited for the rapid and convenient transport of the graft. Therefore, solutions that can autonomously deliver oxygen during graft transport would be beneficial for the improvement of organ transplantation. [0005] To date, several strategies have been trialed to improve the oxygen supply during organ transport. 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 (Ca0₂) have been utilized to deliver oxygen to cells dispersed within a hydrogel matrix.¹⁵ Ca0₂ dispersed within a poly(dimethylsiloxane) (PDMS) polymer matrix was used to deliver oxygen over 40 days to pancreatic β -cells. In this device, water diffuses into the PDMS matrix and reacts with the peroxide, resulting in the formation of oxygen that is then released into the cell suspension in the hydrogel matrix ¹⁵ Whilst the slow and sustained release of oxygen was optimal for this application, aimed at the long-term (weeks) supply of oxygen to pancreatic β-cells after implantation, a more rapid and substantial release of oxygen is required for the short term (hours) transportation of tissues and organs.

[0006] There is thus a need to provide improved substrates and/or compositions that are capable of releasing oxygen into the environment surrounding the substrate, device and/or composition.

SUMMARY

[0007] 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.

[0008] According to a first aspect, there is provided a substrate for sustaining isolated cells, tissues and/or organs by supplying oxygen thereto, 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.

[0009] According to a second aspect, there is provided a substrate for lowering the risk of hypoxia in isolated cells, tissues and/or organs, 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. [0010] According to a third aspect, there is provided 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.

[0011] According to a fourth aspect, there is provided 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.

[0012] According to a fifth aspect, there is provided a 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.

[0013] When 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. When the substrate or composition is contained in a fixed volume the partial pressure of oxygen in the fixed volume then increases.

[0014] In certain embodiments of the first to fifth aspects, the substrate or composition is biocompatible. BRIEF DESCRIPTION OF THE FIGURES

[0015] Embodiments of the present disclosure will be discussed with reference to the accompanying figures wherein:

[0016] Figure 1 shows different embodiments of a substrate of the present disclosure;

[0017] 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; [0018] 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;

[0019] Figure 4 shows (A) Percentage viable MIN6 cells when cultured with oxygen generating substrates containing urea or calcium peroxide capped with one or two outer plasma polymer layers, as measured by resazurin assay. Results are normalised to MIN6 cell cultures not exposed to the oxygen generating substrates (control); error bars show standard deviation for n = 3. (B) Cell viability as a function of ROS generation for samples of different thickness loaded with calcium and urea peroxide, normalized to negative control (0 %): culture media alone and maximum of ROS measured (100%). Each data point represents one measurement for one sample;

[0020] Figure 5 shows (A) dissolved oxygen concentration over time produced in deoxygenated PBS at 20 °C by 21 μιηοΙ/mL of Ca0₂ 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 μιηοΐ) of urea peroxide and coated with two or three outer plasma polymer layers;

[0021] 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 μιη; and

[0022] Figure 7 shows viable cell number measured by resazurin assay for MIN6 cells cultured with urea peroxide oxygen generating substrates with two and three outer plasma polymer layers under (A) normoxia and (B) hypoxia. Results normalized to controls (untreated MIN6). Error bars represent standard error n = 3. Statistical analysis, t-test two tails with * for p < 0.1, ** for p < 0.05 and *** for p < 0.01.

DESCRIPTION OF EMBODIMENTS

[0023] As shown in Figure 1, provided herein is 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. In this case, 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. In this way, water ingress to the oxygen generating material and release of oxygen therefrom only occurs through the water and oxygen permeable membrane and the ingress of water to the oxygen generating material and the rate of release of oxygen from the substrate are controlled by the water and oxygen permeable membrane.

[0024] Thus, provided herein is 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.

[0025] By modulating the diffusion of water through the water and oxygen permeable membrane, oxygen can be released in a controlled fashion into a cell or tissue transport environment to improve cell or tissue preservation. Thus, provided herein is a substrate 10 for sustaining isolated cells, tissues and/or organs by supplying oxygen thereto. The substrate 10 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.

[0026] Also provided herein is a 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.

[0027] Also provided herein is packaging suitable for transporting isolated cells, tissues and/or organs. 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.

[0028] 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. In some embodiments, the substrate may be part of a packaging material and may, for example, be coated onto a known thermoplastic material. In some other embodiments, the substrate may be in the form of a disc, capsule or ball that may be included within packaging. [0029] Substrate materials that can be used include polyolefins, such as polypropylene, polyethylene, polyisoprene, polybutadiene, and polybutene. Other 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.

[0030] 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.

[0031] In certain embodiments, the peroxide is a metal peroxide, such as calcium peroxide (Ca0₂), lithium peroxide (Li₂0₂), sodium peroxide (Na₂0₂), beryllium peroxide (Be0₂), magnesium peroxide (Mg0₂), zinc peroxide (Zn0₂) or copper peroxide (Cu0₂). 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 0₂. Other inorganic peroxides that could be used include percarbonates, such as sodium percarbonate.

[0032] In certain other embodiments, 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 0₂. In the case of urea peroxide, the complexed hydrogen peroxide degrades to give 0₂. 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.

[0033] The oxygen generating material may be a solid, a liquid, a gel, an emulsion, etc

[0034] 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.

[0035] The aqueous liquid can be any liquid that contains water. For example, the aqueous liquid can be water, saline solution, buffer solution, body fluids, water contained in tissues, etc. When the substrates described herein are used to supply oxygen for organ or tissue transportation applications, 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.

[0036] 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. This can be achieved by using a hydrophobic polymer. The hydrophobic polymer may be a polyalkyl or a polyalkenyl homopolymer or copolymer. In certain embodiments, 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"

[0037] In certain embodiments, 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. Other suitable organic compounds may include heteroatoms. One suitable monomer for this purpose is 1,7-octadiene.

[0038] In certain specific embodiments, 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 By varying 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.

[0039] Advantageously, the octadiene plasma polymer is biocompatible. Therefore, in embodiments the substrate or composition is biocompatible.

[0040] In certain embodiments, the oxygen generating substrate comprises oxygen generating material sandwiched between two water and oxygen permeable membrane layers. In these embodiments, the thickness of the oxygen generating substrate is between about 100 nm and about 100 μπι, such as between about 200 nm and about 40μπι. [0041] We have shown that 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. Advantageously, the oxygen generating devices and substrates described herein generate sufficient oxygen to support cell survival under hypoxia, such as about 0.5 % oxygen.

[0042] Many applications of 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). Advantageously, the thickness of the water and oxygen permeable membrane can be used to suppress the release of ROS from the oxygen generating device. In certain embodiments, the oxygen generating devices and substrates described herein generate between 0% and 10% ROS.

[0043] 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). In the case of the oxygen generating substrates described herein, 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.

[0044] Also provided herein is a 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.

EXAMPLES

[0045] Fabrication of oxygen generating substrates

[0046] PDMS disks were prepared from a 1 : 10 w/w precursor mixture of Sylgard® 184 Silicon

Elastomer curing agent and Sylgard® 184 Silicon Elastomer base from Dow (Midland, Michigan, USA). The precursor mixture was placed under vacuum (0.1 mbar) until all the air bubbles were removed, and then 2 g was poured in the lid of a Petri dish and placed at 65 °C in an oven for 4 h. The cured PDMS was removed from the Petri dish and small disks with a diameter of 1.9 cm were punched out with a biopsy tool.

[0047] 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 ^χ 10⁴ mbar using a two-stage rotary vane pump with a liquid N₂ cold trap. To clean and prime the PDMS surfaces, 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. This process ensures that any unwanted organic material on the PDMS surface are removed prior to polymer deposition and that the surface is oxidized to improve adhesion of subsequent layers Subsequently, the pressure in the chamber was lowered to 3.5 ^χ 10^~2 mbar and the monomer, 1,7- octadiene (98 %, Sigma Aldrich), was introduced via a needle valve until steady working flow rates were achieved. The monomer was degassed via three freeze / thaw cycles prior to use. The plasma was ignited with a 50 W CW RF for 5 min to deposit the first plasma polymer layer. Along with the PDMS disks, clean silicon wafers were also exposed to the same treatment for further characterization of the plasma polymer coatings.

[0048] Calcium (75 %, Sigma Aldrich) and urea peroxides (97%, Sigma Aldrich) were milled on a laboratory planetary mill and sieved on a vibratory sieve shaker both from Fritsch (Idar-Oberstein, Germany). Sieving was conducted with a 40 μιη cut off to yield a fine microparticle powder. The peroxide microparticles were then spread across the PDMS disks coated with a base layer of octadiene plasma polymer using a spatula. Subsequently, the disks were reintroduced into the plasma chamber and the top layers of plasma polymer were deposited using the same reactor conditions as described above, for cycles of 20 min to avoid heating of the chamber.

[0049] X-ray photoelectron spectroscopy (XPS). Blank or plasma polymer coated PDMS disks were characterized on a Specs SAGE XPS spectrometer using a Mg K radiation source ( v = 1253.6 eV) operating at 10 kV and 20 mA. 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.

[0050] Contact angle. A custom-built sessile drop apparatus with an Olympus SZ-PT microscope and lens system integrated with a Sony CCD camera was employed to measure the wettability of the plasma coated surfaces. A 10 syringe (Hamilton, Reno, USA) was used to dispense droplets of Milli-Q water (~ 1 μί) on the coated PDMS disks. A minimum of three contact angle measurements were taken from each surface. Angle analysis of captured droplets was performed with Image J software vl .50 with the Drop Snake plugin ⁵

[0051] Ellipsometry. The coating thickness on silicon wafers was determined via ellipsometry using a J. A. Woolam Co. (Lincoln, Nebraska, USA) variable angle spectroscopic ellipsometer (VASE). All measurements and data were analyzed using WVASE32 software provided with the instrument. Three silicon wafers from independent plasma depositions were used for thickness measurements. Polymer thickness values were estimated by applying a Cauchy model. [0052] Oxygen release. Oxygen release from the oxygen generating substrates was measured using a dissolved oxygen probe (Thermo-Fisher). Dissolved oxygen concentrations were reported in mg/L. Measurements were performed in deoxygenated PBS (10 mL) at pH 7.4 in a sealed 23 mL plastic container. 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.

[0053] 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 β- mercaptoethanol solution (5 μΕ/L, Sigma). Once confluent, cells were trypsinized with a solution of 0.05 % trypsin EDTA (Sigma Aldrich).

[0054] 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 χ 10⁵ 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. The well plates were placed in the incubator at 37 °C for 1 h. Finally, the fluorescence was recorded on a Biotek Synergy HT plate reader (Winooski, Vermont). Each sample was tested in triplicate. The results were expressed in percentage of cell survival relative to cells incubated in media in the absence of oxygen generating substrates (100%).

[0055] Quantification of reactive oxygen species (ROS). In order to quantify ROS released from the oxygen generating substrates a 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was employed.³⁶³⁷ DCFH-DA was deacetylated by sodium hydroxide to form non-fluorescent 2,7- dichlorodihydrofluorescein (DCFH). DCFH could then be converted by ROS to the highly fluorescent compound DCF (2,7-dichlorofluorescein). The fluorescence level is proportional to the quantity of ROS. In brief, 2 mM DCFH-DA (500 μί) in water was added to 10 mM sodium hydroxide (2 mL) (BioXtra 98%, Sigma Aldrich). This solution was stored at 4 °C in the dark for 30 min and then added to filtered PBS solution (20 mL) at pH 7.4. 2.5 mL of this solution was added to each well of a 12 well plate containing the oxygen generating substrates to be tested. Each condition was repeated in triplicate. After 4 h, the formation of ROS was measured with a Biotek Synergy HT microtiter plate reader (Winooski, Vermont) at 485 nm excitation and 535 nm emission. The results were expressed in percentage of ROS released from each oxygen generating substrate, with 100 % referring to the sample with the highest level of ROS release and 0 % referring to a control sample with only culture media.

[0056] Hypoxia experiments. Hypoxic conditions were created in a sealed plastic container with Oxoid™ AnaeroGen™ (Thermo Fischer) anaerobic gas generating sachets. MIN6 cells were seeded either under normoxia (21 % 0₂) or hypoxia (0.5 % 0₂) and incubated for 24 h at 37 °C in a 5 % C0₂ 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.

[0057] FDA/PI live dead assay. MIN6 cells were stained with fluorescein diacetate (FDA, Sigma Aldrich) and propidium iodide (PI, Sigma Aldrich). In brief, MIN6 cells were seeded at a density of 2 x 10⁵ cells/mL into a 12 well plate (400 μΐ/ννεΐΐ). PI and FDA solubilized in PBS was added to achieve a final concentration of 5 μg/mL and 5 μΜ, respectively. Samples were incubated for 5-10 min prior to imaging. Fluorescence microscopy images were taken using a Nikon Eclipse TiS microscope.

[0058] Results and discussion

[0059] 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 (Ca0₂) were then manually deposited on the coated PDMS substrates. In order to obtain a homogeneous peroxide layer, the peroxides were mechanically milled and sieved to obtain particles with a size < 40 μιη. 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.

[0060] 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.¹⁵ 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

[0061] Table 1 - Oxygen-generating reagents used in this study and their respective reactions with water.^24,25

Nunc Ty e Formula Oxygen eleasing rea tion

Calcium Peroxide inorganic C.--0 2 C-.0 « 4 Β Ο—^■- 2 CM ) « 2 H O

Urea Peroxide Organic CH.N O 2 ^;-· Q ^■— »2 H O » 0

[0062] Deposition of the octadiene plasma polymer layers that form the oxygen generating substrates were confirmed by X-ray photoelectron spectroscopy (XPS). Upon deposition of a base layer of octadiene plasma polymer, the almost complete reduction of the silicon peaks at 156 and 105 eV confirmed the formation of a complete conformal polymer layer on the PDMS disks (Figure 3A and B). The small Si peaks observed may originate from non-crosslinked polymer chains of PDMS moving toward the surface under the high vacuum of the XPS instrument ( 10^"5 mbar).²⁶

[0063] In order for oxygen to be generated from the peroxide microparticles, decomposition must be triggered by water diffusing across the encapsulating outer plasma polymer layer, as described previously for water soluble drugs.¹⁸ 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 oxygen generating substrates was shown to be governed by the plasma polymer deposition parameters.¹⁸ By varying the thickness of the outer plasma polymer layer, it was possible to control water ingress and the oxygen release rate. The thickness of the outer plasma polymer layer was increased by successively depositing polymer films using the same plasma reactor parameters, thus providing a similar thickness for each deposition. 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.

[0064] One of the challenges of this approach is to only deliver oxygen 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).²⁷ In order to test the efficacy and cytocompatibility of the oxygen generating substrates a model system was chosen that is highly sensitive to oxygen levels and ROS; MIN6 cells, a model for islet cells have a high metabolic rate and are very sensitive to variations in oxygen levels.²⁸ Following culture on the oxygen generating substrates loaded with calcium or urea peroxide and with different outer plasma polymer layer thicknesses for a period of 16 h under normoxic conditions (21 % oxygen) the viable MIN6 cell number was assessed via a resazurin assay (Figure 4A). Oxygen generating substrates loaded with Ca0₂ were found to induce complete cell death independent of the thickness of the plasma polymer coating. In contrast, 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).

[0065] As previously discussed, the observed toxicity may originate from the release of hydrogen peroxide and other ROS into the culture medium.²⁷'^29,3" We therefore measured the ROS concentration within the culture medium for different oxygen generating substrates loaded with either urea or calcium peroxide with one or two outer layers. A plot of ROS concentration against cell viability revealed that the concentration of ROS linearly correlated to the cell viability (Figure 4B).

[0066] These results indicate that the Ca0₂ loaded oxygen generating substrates induce more cell death than urea peroxide due to the release of ROS into the culture media. Additionally, experiments conducted with the urea peroxide loaded oxygen generating substrates indicate that the thickness of the outer layer controls the release of ROS, whereby a single outer plasma polymer layer (~ 40 nm) exhibited higher cell death than two outer layers (~ 90 nm combined thickness) (Figure 4A). Therefore, for the single outer layer hydrogen peroxide is directly diffusing through the octadiene plasma coating without having time to decompose to oxygen. This might be induced by a difference in the oxygen generation kinetics. Indeed, production of oxygen from Ca0₂ microparticles in phosphate buffered saline (PBS) (21 mmol/L taking into account 75 % purity) was slower than that of urea peroxide microparticles (21 mmol/L) (Figure 5A). The decomposition of Ca0₂ into oxygen has been reported to proceed via either a single-step or two-step thermodynamically driven mechanism.³¹ In contrast, urea peroxide decomposition and the production of oxygen follows zero order kinetics ² As a result of the significantly different mechanisms that operate to generate oxygen from the peroxides, a direct comparison is impractical, nevertheless, more ROS and less oxygen are generated with Ca0 microparticles. In the case of the oxygen generating substrates described herein, 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. In order to minimize cell death, the urea peroxide oxygen generating substrates with two or more outer plasma polymer layers (combined thickness > 90 nm) were employed in subsequent experiments.

[0067] To assess the rate of decomposition of urea peroxide and the extent of oxygenation, the amount of dissolved oxygen produced by solubilizing different amounts (0.8, 1.4 and 2.0 mg/mL) of pure urea peroxide in deoxygenated PBS (pH 7.4) in a closed system was measured over 6 h. As a control the concentration of oxygen in the deoxygenated PBS was measured over the same period of time. The amount of dissolved oxygen was found to be approximately proportional to the amount of urea peroxide introduced into solution (Figure 5B). For urea peroxide concentrations of 0.8 and 1.4 mg/mL, a plateau was reached after 100 min and the dissolved oxygen concentration remained constant at ~ 2 and ~ 6 mg/L, respectively. The higher urea peroxide concentration of 2 mg/mL increased the dissolved oxygen concentration to -12 mg/L over 400 min. Subsequently, the dissolved oxygen produced by oxygen generating substrates loaded with 2 mg of urea peroxide with two (~ 90 nm) and three outer plasma polymer layers (-120 nm) was measured over 400 min in PBS ( 10 mL) (Figure 5C). The dissolved oxygen concentration was found to increase linearly over 6 h for the oxygen generating substrate with two outer layers, whereas the concentration increased at a slower rate after 200 min for the oxygen generating substrate with three outer layers. This experiment verified that the thickness of the octadiene plasma polymer film can be used to control the release rate of oxygen from the oxygen generating substrates, as was previously reported for the diffusion of biologically active molecules from plasma polymer coatings.¹⁸

[0068] To demonstrate the potential application of the oxygen generating substrates to reverse hypoxia and improve tissue survival and preservation, cells were incubated under hypoxic conditions in the presence and absence of the oxygen generating substrates. The ability of the oxygen generating substrates to release sufficient oxygen to restore cell viability of hypoxic MIN6 cells was characterized by incubating a MIN6 cell monolayer for 24 h under 0.5 % oxygen and then exposing the cells to oxygen generating substrates loaded with 2 mg of urea peroxide and coated with either two or three outer plasma polymer layers. Firstly, the cell viability was assessed by a live/dead assay (FDA/PI) (Figure 6). MIN6 cells cultured under normoxia (21 % 0₂) did not exhibit any observable cell death (Figure 6A and B), whereas incubation under hypoxia (0.5 % 0₂) for 24 h resulted in nearly complete cell death (Figure 6 C and D). In comparison, when the cells were incubated under hypoxia (0.5 % 0₂) with oxygen generating substrates with either two or three outer plasma polymer layers, cell death appeared to be reduced greatly (Figure 6 E to H). These findings suggest that the oxygen generating substrates can limit the effects of hypoxia on MIN6 cells.

[0069] 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 % 0₂) and hypoxic (0.5 % 0 ) conditions using a resazurin assay (Figure 7). Under normoxic conditions, 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.³³ However, under hypoxia, the MIN6 cell viability increased significantly in the presence of the oxygen generating substrate with two outer plasma polymer layers, with a 300 % increase in cell viability versus the control. Interestingly, the oxygen generating substrate with three outer plasma polymer layers only lead to a very slight increase in cell viability (-10 %) (Figure 7B) even though it was shown previously to increase the dissolved oxygen concentration (Figure 5B). The discrepancy between two and three outer layers may originate from the release rate of oxygen being slower and insufficient for the oxygen generating substrate with three outer layers (Figure 5B). These results indicate that the oxygen generating substrate with two outer plasma polymer layers is optimal and can significantly improve cell survival under hypoxic conditions.

[0070] Conclusion

[0071] We have demonstrated that thin biocompatible plasma polymer films can be utilized to encapsulate oxygen generating agents to afford oxygen generating substrates. This technique was used to coat PDMS disks, but can potentially be applied to a broad range of surfaces. Upon contact with aqueous solutions, the oxygen generating substrates were capable of increasing the dissolved oxygen

concentration. The plasma polymer coating thickness and choice of oxygen-generating agent play a role in reducing cell toxicity and especially the generation of ROS. Finally, we showed that urea peroxide - based oxygen generating substrates were able to restore MIN6 cell viability under hypoxic conditions. 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.

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[00109] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[00110] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge. [00111] It will be appreciated by those skilled in the art that the invention is not restricted in its use to the particular application described. Neither is the present invention restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the invention is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

Claims

1. A substrate for sustaining isolated cells, tissues and/or organs by supplying oxygen thereto, 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.

2. A substrate for lowering the risk of hypoxia in isolated cells, tissues and/or organs, 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.

3. 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.

4. The substrate according to any one of the preceding claims, wherein the substrate is formed from a material selected from one or more of the group consisting of polyolefins, fluorinated polymers, polysiloxanes, polycarbonates, polyamides, ethylene -vinyl acetate copolymers, ethylene- methacrylate copolymers, poly(vinyl chloride), polystyrene, polyesters, polyanhydrides, polyacrylianitrile, polysulfones, polyacrylic ester, acrylic, polyurethane, polyacetal, and copolymers or mixtures thereof.

5. The substrate according to any one of the preceding claims, wherein the oxygen generating material is a substance capable of degrading to release hydrogen peroxide or any substance containing hydrogen peroxide which then degrades further to release oxygen.

6. The substrate according to claim 5, wherein the oxygen generating material is a peroxide.

7. The substrate according to claim 6, wherein the peroxide is a metal peroxide.

8. The substrate according to claim 7, wherein the metal peroxide is selected from one or more of the group consisting of calcium peroxide (Ca0₂), lithium peroxide (Li₂0₂), sodium peroxide (Na₂0₂), beryllium peroxide (Be0₂), magnesium peroxide (Mg0₂), zinc peroxide (Zn0₂) and copper peroxide (Cu0 ). The metal peroxide could also be any suitable peroxide of a group I or group II metal.

9. The substrate according to claim 6, wherein the peroxide is a percarbonate.

10. The substrate according to claim 9, wherein the percarbonate is sodium percarbonate.

11. The substrate according to claim 6, wherein the peroxide is a non-metallic peroxide complex.

12. The substrate according to claim 11, wherein the non-metallic peroxide complex is selected from one or more of the group consisting of urea peroxide and ammonium peroxide.

13. The substrate according to any one of the preceding claims, wherein the oxygen generating material is a neat material.

14. The substrate according to any one of claims 1 to 12, wherein the oxygen generating material is deposited on a sponge-like or open-celled foam support material.

15. The substrate according to any one of the preceding claims, wherein 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.

16. The substrate according to claim 15, wherein the water and oxygen permeable membrane is a hydrophobic polymer.

17. The substrate according to claim 16, wherein the hydrophobic polymer is a polyalkyl or a polyalkenyl homopolymer or copolymer.

18. The substrate according to any one of claims 15 to 17, wherein the polymer is a plasma polymer.

19. The substrate according to any one of claims 15 to 18, wherein the water and oxygen permeable membrane is plasma polymerized 1,7 octadiene.

20. The substrate according to any one of the preceding claims, wherein the thickness of the water and oxygen permeable membrane is from about lOnm to about 150nm.

21. The substrate according to claim 20, wherein the thickness of the water and oxygen permeable membrane is about 40 nm, about 80 nm or about 120 nm.

22. The substrate according to any one of the preceding claims, wherein the substrate comprises oxygen generating material sandwiched between two water and oxygen permeable membrane layers.

23. The substrate according to claim 22, wherein the thickness of the oxygen generating substrate is between about 100 nm and about 100 μιη.

24. The substrate according to claim 23, wherein the thickness of the oxygen generating substrate is between about 200 nm and about 40μιη.

25. The substrate according to any one of the preceding claims, wherein the substrate generates sufficient oxygen to support cell survival under hypoxia.

26. The substrate according to claim 25, wherein the substrate generates about 0.5 % oxygen.

27. The substrate according to any one of the preceding claims, wherein the substrate generates between 0% and 10% reactive oxygen species.

28. 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.

29. A 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.