Blood preservation compositions, devices and uses thereof utilizing: a. zirconium dioxide nanoparticles or nanoporous zirconium dioxide nanoparticle macrostructures; and b. nanoporous cerium oxide nanoparticle macrostructures

Abstract

Blood preservation compositions for blood and blood products, devices and uses thereof that utilize zirconium dioxide (ZrO2) nanoparticles and / or nanoporous zirconium dioxide nanoparticle (NZrO2NP) macrostructures and nanoporous cerium dioxide nanoparticle (NCeONP) macrostructures. The blood preservation compositions are particularly suitable to increase the shelf life of blood and blood products which includes reductions in oxidative and storage hemolysis.

Description

[0001] Blood Preservation Compositions, Devices And Uses Thereof Utilizing:

[0002] A. Zirconium Dioxide Nanoparticles or Nanoporous Zirconium Dioxide Nanoparticle Macrostructures; And

[0003] B. Nanoporous Cerium Oxide Nanoparticle Macrostructures

[0004] Cross-Reference To Related Applications

[0005] This application claims the benefit of U.S. Provisional Application Ser. No. 63 / 253,629 filed October 8, 2021, which is fully incorporated by reference.

[0006] Field

[0007] The present invention is directed at blood preservation compositions for blood and blood products, devices and uses thereof that utilize zirconium dioxide (ZrO2) nanoparticles and / or nanoporous zirconium dioxide nanoparticle (NZrO2NP) macrostructures and nanoporous cerium dioxide nanoparticle (NCeONP) macrostructures. The blood preservation compositions are particularly suitable to increase the shelf life of blood and blood products and to provide a blood bag with adequate storage capability that avoids the use for phthalate type plasticizers, such as di(2-ethylhexyl phthalate).

[0008] Background

[0009] Storage of blood, blood products such as RBC, platelets etc., and components thereof is important for many clinical purposes. Current blood storage method can be time limited, which can lead to unusable blood, blood product and components thereof, particularly where relatively lengthy and / or unrefrigerated storage is needed. Blood bags utilizing phthalate plasticizers, such as di(2-ethylhexyl phthalate) or DEHP, have been successful as DEHP is a useful plasticizer. However, DEHP can have an adverse effect on blood storage hemolysis and oxidative hemolysis. PCT / US2019 / 043785 reports on blood storage containers that contain a cerium oxide nanoparticle (CeONP) composition and / or coating. The CeONP compositions and / or coating on the object surface can be effective to increase the useful storage lifespan of blood, blood product and / or component thereof stored in the blood storage container. Accordingly, there remains an on-going need for improved and / or additional compositions and techniques to store blood, blood products and or components thereof. In particular, compositions that allow for the replacement of phthalate plasticizers in blood bags, such as DEHP, that provide adequate storage capability. Summary A blood storage container comprising a blood storage surface comprising zirconium dioxide (ZrO2) nanoparticles and / or nanoporous zirconium dioxide nanoparticle (NZrO2NP) macrostructures, present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, and combinations thereof when such blood, blood product(s) and components thereof are stored in contact with the blood storage container surface. A method of storing whole blood, blood product(s), components thereof or combinations thereof, comprising: supplying a blood storage container comprising a blood storage surface comprising zirconium dioxide (ZrO2) nanoparticles and / or nanoporous zirconium dioxide nanoparticle (NZrO2NP) macrostructures present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, or combinations thereof when such blood, blood product(s) and components thereof are stored in contact with the blood storage container surface. This is followed by storing blood, blood product(s), components thereof or combinations thereof in the blood storage container.

[0010] A method of storing blood, blood product products(s), components therefor or combination thereof comprising: supplying blood, blood product(s) or components thereof; combining the blood, blood product(s) or components thereof with zirconium oxide nanoparticles and / or nanoporous zirconium oxide nanoparticle macrostructures wherein the concentration of the zirconium oxide nanoparticles and / or nanoporous zirconium oxide nanoparticle macrostructures is in the range of 1 pM to IM.

[0011] A blood container insert having a surface, comprising a coating, wherein the coating comprises zirconium dioxide (ZrO2) nanoparticles and / or nanoporous zirconium dioxide nanoparticle macrostructures in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, or combinations thereof, when such blood, blood product(s) and components thereof are stored in contact with the insert inside of the blood storage container.

[0012] A blood storage container comprising a blood storage surface comprising: nanoporous cerium oxide nanoparticle (NCeONP) macrostructures present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, and combinations thereof when such blood, blood product(s) and components thereof are stored in contact with the blood storage container surface.

[0013] A method of storing whole blood, blood product(s), components thereof or combinations thereof, comprising: (a) supplying a blood storage container comprising a blood storage surface comprising nanoporous cerium oxide nanoparticle macrostructures present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, or combinations thereof when such blood, blood product(s) and components thereof are stored in contact with the blood storage container surface; (b) storing blood, blood product(s), components thereof or combinations thereof in said blood storage container.

[0014] A blood container insert having a surface, comprising a coating, wherein the coating comprises nanoporous cerium oxide nanoparticle macrostructures in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, or combinations thereof, when such blood, blood product(s) and components thereof are stored in contact with the insert inside of the blood storage container.

[0015] A method of storing blood, blood product products(s), components therefor or combination thereof comprising: supplying blood, blood product(s) or components thereof; combining the blood, blood product(s) or components thereof with nanoporous cerium oxide nanoparticle macrostructures wherein the concentration of the nanoporous cerium oxide nanoparticle macrostructures is in the range of 1 pM to IM

[0016] Drawings

[0017] FIG. 1 demonstrates the protective effect of ZrO2nanoparticles with diameters of 20 nm to 30 nm against AAPH-induced oxidative hemolysis in whole blood stored in the presence of ZrO2nanoparticles at the indicated concentrations (0-1000 nM) over the indicated time periods (0- 14 days).

[0018] FIG. 2 demonstrates the protective effect of ZrO2nanoparticles with diameters of <5.0 nm against AAPH-induced oxidative hemolysis in whole blood stored in the presence of ZrO2 nanoparticles at the indicated concentrations (0-1000 nM) over the indicated time periods (0-14 days).

[0019] FIG. 3 demonstrates the protective effect of ZrO2nanoparticles with diameters of 20.0 nm to 30.0 nm against AAPH-induced oxidative hemolysis in RBC concentrates stored in the presence of ZrO2nanoparticles at the indicated concentrations (0-1000 nM) over the indicated time periods (0-14 days).

[0020] FIG. 4 illustrates the percent storage hemolysis for ZrO2nanoparticles with diameters of 20.0 nm to 30.0 nm at the indicated concentrations (0-1000 nM) and over the indicated time periods (0-14 days) showing its protective effective against storage hemolysis in RBC concentrates.

[0021] FIG. 5 illustrates the percent osmotic hemolysis for the ZrO2nanoparticles having diameters in the range of 20.0 nm to 30.0 nm at the indicated concentrations (0-1000 nM) in whole blood and over the indicated time periods (0-14 days) showing its protective effect against osmotic hemolysis

[0022] FIG. 6 illustrates the nanoporous zirconium oxide nanoparticle (NZrO2NP) macrostructure sourced from zirconium oxide nanoparticles.

[0023] FIG. 7 illustrates the percentage of AAPH-induced oxidative hemolysis of whole blood for the ZrO2nanoparticles having diameters in the range of 20 nm to 30 nm at the indicated concentrations (0-100 pM) over the indicated time periods (0-28 days) showing its protective effective against oxidative hemolysis in whole blood.

[0024] FIG. 8 illustrates the percentage oxidative hemolysis in whole blood for the ZrO2nanoparticles having diameters less than 5 nm at the indicated concentrations (0-100pM) over the indicated time periods (0-28 days) showing its protective effect against oxidative hemolysis in whole blood.

[0025] FIG. 9 illustrates the nanoporous cerium oxide nanoparticle (NCeONP) macrostructure sourced from cerium oxide.

[0026] FIG. 10 illustrates the % AAPH-induced oxidative hemolysis in whole blood for the NCeONP macrostructures at the indicated concentrations (0-100 μM) over the indicated time periods (0-28 days) demonstrating its protective effect against oxidative hemolysis.

[0027] FIG. 11 illustrates the % AAPH-induced oxidative hemolysis of whole blood for the NCeONP macrostructures at the indicated concentrations (0-1000 nM) over the indicated time periods (0-14 days) demonstrating its protective effect against oxidative hemolysis of whole blood.

[0028] FIG. 12 illustrates the % hemolysis in RBC concentrates for the NCeONP macrostructures at the indicated concentrations (0-1000 nM) over the indicated time periods (0-14 days) demonstrating its protective effect against oxidative hemolysis of RBC concentrates.

[0029] FIG. 13A illustrates the efficacy of a 50% mixture by weight of zirconium oxide nanoparticles and nanoporous cerium oxide macrostructures in preventing oxidative hemolysis in whole blood (WB).

[0030] FIG. 13B illustrates the efficacy of a 50% mixture by weight of zirconium oxide nanoparticles and nanoporous cerium oxide macrostructures in preventing oxidative hemolysis in RBCs. FIG. 14 illustrates the oxidative hemolysis versus storage week time of NCeONP macrostructures or ZrC>2 nanoparticles in a PVC film insert when inserted into an X-ray irradiated PVC blood bag made using DEHP plasticizer.

[0031] FIG. 15 illustrates the percent methemogloblobin (oxidized hemoglobin) versus storage week time of NCeONP macrostructures or ZrO2nanoparticles in a PVC film insert containing DEHT when inserted into a commercial PVC blood bag (x-ray irradiated) made using DEHP plasticizer.

[0032] FIG. 16 illustrates red cell distribution width (RDW-CV) versus storage week time of NCeONP macrostructures or ZrO2nanoparticles in a PVC film insert containing DEHT when inserted into a commercial PVC blood bag (x-ray irradiated) made using DEHP plasticizer.

[0033] FIG. 17 illustrates glucose (mmol / L) versus storage week time of NCeONP macrostructures or Z1O2nanoparticles in a PVC film insert containing DEHT when inserted into a commercial PVC blood bag (x-ray irradiated) made using DEHP plasticizer.

[0034] FIG. 18 illustrates lactate (mmol / L) versus storage week time of NCeONP macrostructures or ZrO2nanoparticles in a PVC film insert containing DEHT when inserted into a commercial PVC blood bag (x-ray irradiated) made using DEHP plasticizer.

[0035] FIG. 19 illustrates percent oxygen saturation versus storage week time of NCeONP macrostructures or ZrO2nanoparticles in a PVC film insert containing DEHT when inserted into a commercial PVC blood bag (x-ray irradiated) made using DEHP plasticizer.

[0036] FIG. 20 illustrates the percent storage hemolysis of RBCs in a commercial blood bag made with DEHP plasticizer versus storage week time, with PVC film insert containing a 2.0% (wt.) loading of NCeONP macrostructures and DEHT plasticizer. FIG. 21 shows the storage hemolysis of RBCs versus storage week time for a blood bag (non-irradiated) made of PVC plasticized with DEHT and containing 5.0 % (wt.) of NCeONP macrostructures. Also shown is the European Union limit.

[0037] FIG. 22 shows the percent oxidative hemolysis of RBCs versus storage week time for a blood bag (non-irradiated) made of PVC plasticized with DEHT and containing 5.0 % (wt.) of NCeONP macrostructures.

[0038] Detailed Description Of Preferred Embodiments

[0039] The present invention is directed at blood preservation compositions that comprise zirconium dioxide (ZrO2) nanoparticles and / or nanoporous zirconium oxide nanoparticle (NZrO2NP) macro-structures containing a plurality of zirconium oxide nanoparticles which define a plurality of macro- structure pores. The ZrO2nanoparticles and / or NZrOzNP macro-structures are present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, when such blood, blood product(s) and components thereof are in contact with the ZrO2nanoparticles and / or NZrO2NP macro-structures.

[0040] The above referenced zirconium dioxide nanoparticles are preferably degassed with nitrogen for a preferred period of 30 minutes or 60 minutes. It is contemplated that this then can be followed by heating at elevated temperatures, preferably in the range of 50 °C to 900 °C for a preferred period of 1.0 hour to 3.0 hours, more preferably 1.0 hour to 2.0 hours. Accordingly, it is contemplated that such heating of the zirconium oxide nanoparticles will form a plurality of nanoporous zirconium oxide nanoparticle macrostructures 12 illustrated in FIG. 6 having macrostructure pores 14. The macro-structure pores 14 that are formed by the zirconium oxide nanoparticle macrostructure 12 preferably have a diameter (largest linear dimension) as indicated by arrow 15 in the range of 1.0 nm nm to 1000 nm, more preferably, 1 nm to 750 nm or 1 nm to 500 nm or 1 nm to 250 nm or 1 nm to 100 nm or 1 nm to 50 nm or 1 nm to 25 nm. In addition, the nanoporous cerium oxide nanoparticle macro-structures 12 themselves are contemplated to have a preferred diameter (largest linear dimension) as indicated by arrow 16 in the range of 50 nm to 30,000 nm.

[0041] The ZrO2nanoparticles and / or NZrCbNP macro-structures may therefore preferably be included in stored blood, included as a solution in a blood storage container, form a coating on one or more inside surfaces of a blood storage container, or made into films obtained through compounding of the ZrO2nanoparticles and / or NZrCTNP macrostructures with a polymer, via a separate compounding step or via extrusion compounding. Such films may then serve as the insert into the blood storage container or as the film for manufacture of the blood storage container. The ZrO2nanoparticles and / or NZrO2NP macro-structures may also be coated on the surface of an insert that can be included in the blood storage container. The coating is preferably biodegradable, which is reference to the feature that the coating is a material that will degrade under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated or excreted by the subject. The coating is also preferably present at a thickness of 0.1 nm to 100 pm.

[0042] The films containing the ZrO2nanoparticles and / or ZrO2macrostructures or the blood storage container herein may be preferably formed from a polymeric resin and may be in film form, which film may preferably have a thickness from 0.10 mm to 1.00 mm. Preferably, one or more of the following polymers: polyethylene, polypropylene, polybutene, poly(l -octene), a polyester, a polyamide, a polyurethane, a polyurea, a polycarbonate, a polyether, a polyketone, poly(vinyl chloride), a fluoropolymer, or a blend or copolymer thereof or a poly(ethylene-co- propylene), a poly(ethylene-co-methyl acrylate), a poly(ethylene-co-ethyl-acrylate), a low-density polyethylene, a linear low-density polyethylene, or a high-density polyethylene or modified polyolefin comprising polyethylene or polypropylene and the acid anhydride is maleic anhydride. These could also include biodegradable polymers, biopolymers, natural polymers and those made using plant sources. Other polymers include ethylene-vinyl acetate resin, poly siloxane, polycarbonate, polyetheretherketone (PEEK), polyethyleneimine (PEI), polysulofone, and polyurethane resins.

[0043] The ZrO2nanoparticles and / or NZrO2NP macro-structures are therefore preferably in a dispersion as a liquid composition within a blood storage container and capable of mixing with blood, blood product or components thereof stored within the blood storage container. The ZrO2nanoparticles and / or NZrCTNP macro- structures may also be contained in a solid composition (e.g. polymer resin) present within the walls of the blood storage container or within an insert for the container. In that regard, it is contemplated that the surfaces of the container or insert, or surfaces of the film used to make a container or insert, made of polymeric resin, can be made porous, to ensure that the ZrO2nanoparticles and / or the NZrO2NP macro-structures may come in contact with the blood, but otherwise remain immobilized in the polymer resin and not directly mix with the blood contained therein. Similarly, if the ZrO2nanoparticles and / or NZrCTNP macro-structures are contained in a coating on the inside storage surface of a blood storage container or insert, the coating is contemplated to be porous such that the ZrO2nanoparticles and / or the NZrO2NP macro- structures may come in contact with the blood, but again, remains immobilized in the coating.

[0044] For example, the ZrO2nanoparticles and / or NZrO2NP macro- structures may be preferably mixed (blended and dispersed) with PVC or other polymers that can be converted into films that are utilized in a blood bag. It is also contemplated that the PVC or other polymeric film material may be spray coated with the ZrO2nanoparticles and / or NZrO2NP macro- structures dispersed in a liquid medium and the coated films may then be employed in a bag to contain blood. Polymers other than PVC are contemplated herein for formation of film inserts

[0045] The ZrO2nanoparticles and / or NZrO2NP macro-structures are preferably present in the polymer material, at a concentration of 1-10 wt. %. More preferably, when the ZrO nanoparticles and / or NZrO2NP macro-structures are present in a coating, they are present at a preferred concentration of 1-5 wt. %. When the ZrO2nanoparticles and / or NZrO2NP macro- structures are contained in the walls of a polymeric container for blood storage, they are preferably present at a concentration of 1-5 wt. %.

[0046] The ZrO2nanoparticles preferably have a diameter (largest dimension) in the range of 1.0 nm to 1000.0 nm. More preferably, the ZrO2nanoparticles have a diameter in the range of 1.0 nm to 40.0 nm, or 1.0 nm to 30 nm, or 1.0 nm to 20 nm, or 1.0 nm to 10 nm, or 1.0 nm to 5.0 nm.

[0047] The NZrO2NP macrostructures therefore preferably comprise a plurality of zirconium oxide nanoparticles having a diameter in the range of 1.0 nm to 1000.0 nm present as a macrostructure having a macrostructure diameter in the range of 50 nm to 30,000 nm and a macrostructure pore diameter in the range of 4 nm to 1000 nm.

[0048] The concentration of the ZrO2nanoparticles and / or NZrCVNP macro-structures in the blood is preferably established in the range of 1 pM to IM. More preferably, the concentration of ZrO2nanoparticles in the blood is in the range of 1.0 nM to 2000 nM, or 1.0 nM to 1000 nM, or 1.0 nM to 500 nM, or 1.0 nM to 250 nM, or 1.0 nM to to 100 nM, or 1.0 nm to 10.0 nM. It is contemplated that the ZrCT nanoparticles and / or the NZrO2NP macrostructures can be effective to increase the usable storage time of blood, blood product and / or component thereof that is contained within a blood storage container by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.

[0049] It is also contemplated that the ZrO2nanoparticles and / or NZrCTNP macro-structures, in combination with cerium oxide nanoparticles or as alloys of Al, Ta, Mg Zn, Sr, or any other metals or metal oxides can be effective to increase the usable storage time of blood, blood product and / or component thereof that is contained within a blood storage container by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In addition, the ZrO2nanoparticles and / or NZrO2NP macro- structures herein are preferably the primary and / or sole component to provide for an increase the useful storage life of blood, blood product(s), components thereof. That is, the blood storage container of polymeric film inside surface herein is preferably one that consists essentially of and / or consists of, ZrO2nanoparticles and / or NZrCTNP macro-structures in an amount effective to increase the useful storage life of blood, blood product(s), and components thereof, when such blood, blood product(s) and components therefore are stored in contact with the blood storage container inside surface. Reference to blood herein may be understood as reference to whole blood. The term blood product refers to any therapeutic product produced from or with blood as a component thereof. Examples of blood products include red blood cell concentrates, platelets produced from whole blood, plasma, and cryoprecipitates (frozen blood product prepared from blood plasma). The term blood component refers to any part, fraction, cell or molecule of blood.

[0050] The performance of the ZrO2nanoparticles was evaluated by testing for changes in red blood cell susceptibility to selected hemolysis stress tests including oxidative, osmotic, and spontaneous (cold storage) following cold storage (4°C) of whole blood was for 1, 14, and 28 days. Percent oxidative hemolysis was induced by incubating nanoparticle-treated red blood cells (RBCs) and control (no nanoparticles) RBCs with 2,2’-azobis-2-methylpropanimidamide dihydrochloride (AAPH; 150 mmol / L). Thermal (37 °C) decomposition of AAPH generates peroxyl radicals leading to lipid peroxidation-mediated hemolysis. Washed RBCs were suspended with phosphate buffered saline (PBS) to a final concentration of 3.5% + 0.5%. Aliquots (0.21 mL) were transferred into microplates to which 0.09 mL of AAPH (0.5 M) or PBS was added. The plates were incubated (37°C) under static conditions for 1.5 hours, after which the plates were centrifuged (1500g, 10 minutes, 18°C), and AAPH-induced oxidative hemolysis was determined by using the following formula:

[0051] Note that HbAAPH corresponds to supernatant cell-free hemoglobin (Hb) of AAPH-treated RBCs, Hbcontrolcorresponds to supernatant cell-free hemoglobin from untreated RBCs, and Hbtotalrefers to the total amount of hemoglobin in each sample.

[0052] Reference is next made to FIG. 1 which illustrates the percent of oxidative hemolysis of whole blood upon exposure to ZrO2nanoparticles with diameters of 20.0 nm to 30.0 nm at the indicated concentrations and over the indicated time periods. FIG. 2 illustrates the percent of oxidative hemolysis of whole blood upon exposure to ZrO2nanoparticles with diameters of < 5.0 nm at the indicated concentrations over the indicated time periods. As can be observed, the ZrO2nanoparticles are observed to reduce the % Hemolysis, relative to a control, at periods of up to at least 14 days.

[0053] Evaluation of the ZrO2nanoparticles herein was also considered in connection with oxidative hemolysis in RBC concentrates. Results appear in FIG. 3, which identifies the percent of oxidative hemolysis for ZrO2nanoparticles with diameters of 20.0 nm to 30.0 nm at the indicated concentrations and over the indicated time periods. It is again observed that the ZrO2nanoparticles reduce the % oxidative hemolysis, relative to a control, at periods of up to at least 14 days.

[0054] Next, the storage hemolysis was evaluated in red blood cell concentrates. More specifically, percent storage hemolysis is the spontaneous hemolysis that occurs during cold storage (1-6°C) of whole blood bags or RBC concentrates. Percent storage hemolysis was determined at select time points (1, 14, and 30 days) according to the following equation:

[0055] Sample hematocrit (HCT) was determined by collecting blood samples into capillary tubes, which were centrifuged in a micro-HCT centrifuge. Hbsupenatantrefers to the levels of free hemoglobin obtained after centrifugation (1500g, 10 minutes, 18°C) measured in the supernatant. Hbtotalrefers to the total amount of sample hemoglobin before centrifugation.

[0056] Attention is therefore directed to FIG. 4, which illustrates the percent storage hemolysis for ZrO2nanoparticles with diameters of 20.0 nm to 30.0 nm at the indicated concentrations and over the indicated time periods. It is observed that the ZrO2nanoparticles reduce the % storage hemolysis, relative to a control, at periods of up to at least 14 days.

[0057] In addition, an evaluation was made of osmotic hemolysis in red blood cell concentrates. More specifically, percent osmotic hemolysis (also known as osmotic fragility) was determined by incubating (4 hours at 22°C) nanoparticle-treated RBCs and control (no nanoparticles) RBCs in a modified pink test buffer (a hypotonic Bis-Tris buffer containing 25 mmol / L sodium chloride, 70 mmol / L 2,2 bis(hydroxymethyl)-2,2’,2’ nitrilotriethanol (Bis-Tris) buffer, and 135 mmol / L glycerol; pH 6.6) at a final concentration of 1.6% ± 0.2% after which blood samples were centrifuged (1500g, 10 minutes, 18°C), and percent osmotic hemolysis was determined by:

[0058] Note that Hbosmoticcorresponds to supernatant cell-free hemoglobin of pink test-treated RBCs, and Hbtotai refers to the total amount of hemoglobin in each sample.

[0059] Attention is directed to FIG. 5 which illustrates the % osmotic hemolysis for the Z1O2 nanoparticles having diameters in the range of 20.0 nm to 30.0 nm at the indicated concentrations and over the indicated time periods. As can be seen, the ZrCh nanoparticles reduce the % osmotic hemolysis, relative to a control, at periods of up to at least 14 days.

[0060] Attention is next directed to FIG. 7, which illustrates the percentage oxidative hemolysis of whole blood for the ZrO2nanoparticles having diameters in the range of 20 nm to 30 nm at the indicated concentrations over the indicated time periods. Attention is also directed to FIG. 8 which illustrates the percent oxidative hemolysis in whole blood for the ZrO2nanoparticles having diameters less than 5 nm at the indicated concentrations over the indicated time periods. As can be observed, the effect was more pronounced for the ZrO2nanoparticles having diameters in the range of 20 nm to 30 nm.

[0061] As also noted above, the present invention also stands directed at nanoporous cerium oxide nanoparticle (NCeONP) macrostructures for blood preservation. That is, in the above description of ZrO2nanoparticles and / or NZrO2NP macrostructures, one may replace and utilize a nanoporous cerium oxide nanoparticle macrostructure. The NCeONP macrostructures are reference to a plurality of cerium oxide nanoparticles having a diameter in the range of 10 nm to 100 nm present as a macro- structure having macro-structure diameter in the range of 50 nm to 30,000 nm and macro- structure pore diameter in the range of 10 nm to 1100 nm.

[0062] The NCeONP macro-structures may therefore preferably be included in stored blood, included as a solution in a blood storage container, form a coating on one or more inside surfaces of a blood storage container, or made into films obtained through compounding of the NCeONP macrostructures with a polymer, via a separate compounding step or via extrusion compounding. Such films may then serve as the insert into the blood storage container or as the film for manufacture of the blood storage container. The NCeONP macro- structures may also be coated on the surface of an insert that can be included in the blood storage container. The coating is preferably biodegradable, which is reference to the feature that the coating is a material that will degrade under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated or excreted by the subject. The coating is also preferably present at a thickness of 0.1 nm to 100 pm.

[0063] The nanoporoous cerium oxide macro- structures are preferably present in the polymer material at a concentration of 1-10 wt.%. More preferably, when the nanoporous cerium oxide macro- structures are present in a coating, they are present at a preferred concentration of 1-5 wt.%. When the nanoporous cerium oxide macro-structures are contained in the walls of a polymeric container for blood storage, or in a polymeric film insert, they are preferably present at a concentration of 1-5 wt. %.

[0064] In that regard, it is contemplated that the surfaces of the container or insert, or surfaces of the film used to make a container or insert, made of polymeric resin, can be made porous, to ensure that the NCeONP macro-structures may come in contact with the blood, but otherwise remain immobilized in the polymer resin and not directly mix with the blood contained therein. Similarly, if the NCeONP macro-structures are contained in a coating on the inside storage surface of a blood storage container or insert, the coating is contemplated to be porous such that the NCeONP macro- structures may come in contact with the blood, but again, remains immobilized in the coating.

[0065] The films herein containing the NCeONP macrostructures, which can be used for a film insert into a blood storage container, or used directly to form the blood storage container, may preferably have a thickness of 0.10 mm to 1.00 mm. Preferred polymers again include one or more of the following: polyethylene, polypropylene, polybutene, poly(l -octene), a polyester, a polyamide, a polyurethane, a polyurea, a polycarbonate, a polyether, a polyketone, poly(vinyl chloride), a fluoropolymer, or a blend or copolymer thereof or a poly(ethylene-co-propylene), a poly(ethylene-co-methyl acrylate), a poly(ethylene-co-ethyl-acrylate), a low-density polyethylene, a linear low-density polyethylene, or a high-density polyethylene or modified polyolefin comprising polyethylene or polypropylene and the acid anhydride is maleic anhydride. These could also include biodegradable polymers, biopolymers, natural polymers and those made using plant sources. Other polymers include ethylene-vinyl acetate resin, polysiloxane, polycarbonate, polyetheretherketone (PEEK), polyethyleneimine (PEI), polysulofone, and polyurethane resins.

[0066] The concentration of the nanoporous cerium oxide macrostructures in the blood is preferably established in the range of 1 pM to IM (1 mole). More preferably, the concentration of the nanoporous cerium oxide macrostructures in the blood is in the range of 1.0 micro molar (nM) to 1000 micro molar (mM), or 1.0 nM to 2000 nM, or 1.0 nM to 1000 nM, or 1.0 nM to 500 nM, or 1.0 nM to 250 nM, or 1.0 nM to to 100 nM, or 1.0 nm to 10.0 nM.

[0067] The NCeONP macrostructures are preferably prepared by starting with cerium oxide nanoparticles that preferably have a diameter in the range of 10 nm to 100 nm. More preferably, the cerium oxide oxide nanoparticles employed herein have a diameter in the range of 10 nm to 50 nm or 10 nm to 30 nm or 20 nm to 30 nm.

[0068] The above referenced cerium oxide nanoparticles are then preferably degassed with nitrogen for a preferred period of 30 minutes to 60 minutes. This is then preferably followed by heating at elevated temperature, and preferably at the temperature range of 50 °C to 900 °C for a preferred period of 1.0 hour to 3.0 hours, more preferably 1.0 hour to 2.0 hours. Accordingly, such heating of the cerium oxide nanoparticles was observed to form a plurality of nanoporous cerium oxide nanoparticle macro-structures 18 illustrated in FIG. 9 having macro- structure pores 20.

[0069] The macro-structure pores 20 that are formed by the cerium oxide nanoparticle macrostructure 18 preferably have a diameter (largest linear dimension) as indicated by arrow 22 in the range of 10 nm to 1100 nm, more preferably, 10 nm to 750 nm or 10 nm to 500 nm or 10 nm to 250 nm or 10 nm to 100 nm or 10 nm to 50 nm or 10 nm to 25 nm. In addition, the nanoporous cerium oxide nanoparticle macro- structures 18 themselves are contemplated to have a preferred diameter (largest linear dimension) as indicated by arrow 24 in the range of 50 nm to 30,000 nm.

[0070] FIG. 10 illustrates the % oxidative hemolysis in whole blood for the NCeONP macrostructures at the indicated concentrations (0-100 mM) over the indicated time periods (0-28 days) demonstrating its protective effect against oxidative hemolysis. FIG. 11 illustrates the % oxidative hemolysis of whole blood for the NCeONP macrostructures at the indicated concentrations (0-1000 nM) over the indicated time periods (0-14 days) demonstrating its protective effect against oxidative hemolysis of whole blood. FIG. 12 illustrates the % hemolysis in RBC concentrates for the NCeONP macrostructures at the indicated concentrations (0-1000 nM) over the indicated time periods (0-14 days). Fig. 13A illustrates the efficacy of a 50% (wt.) mixture of zirconium oxide nanoparticles and nanoporous cerium oxide macrostructures in preventing oxidative hemolysis in whole blood (WB). The particle size of the zirconium oxide particles was 20 nm to 30 nm and the nanoparticle macro- structures comprised a plurality of cerium oxide nanoparticles having a diameter in the range of 10 nm to 100 nm present as a macro structure having macro structure diameter in the range of 50 nm to 30,000 nm and macro-structure pore size in the range of 10 nm to 1100 nm.

[0071] FIG. 13B illustrates the efficacy of a 50% (wt.) mixture of zirconium oxide nanoparticles and nanoporous cerium oxide macrostructures in preventing oxidative hemolysis in RBCs. The particle size of the zirconium oxide particles was 20 nm to 30 nm and the nanoparticle macro- structures comprised a plurality of cerium oxide nanoparticles having a diameter in the range of 10 nm to 100 nm present as a macro structure having macrostructure diameter in the range of 50 nm to 30,000 nm and macro-structure pore size in the range of 10 nm to 1100 nm. It is therefore contemplated that one may utilize 0. l%-99.9% by weight of zirconium oxide nanoparticles and 99.9%-0.1% by weight of nanoporous cerium oxide macrostructures to improve the storage of blood, blood product(s), or components thereof.

[0072] It is also worth noting that the use of the ZrO2nanoparticles and / or nanoporous zirconimum oxide macro structure and / or cerium oxide nanoporous macro structures herein, either directly in blood, or in the polymeric material to form the blood bag, or as a coating on a polymeric film insert, is contemplated to be particularly applicable in blood bags that avoid the use of phthalate plasticizers, such as DEHP. Accordingly, such blood bags are those that utilize alternatives to phthalate plasticizers such as DEHP, and instead utilize di(2-ethylhexyl) terephthalate (DEHT). Other blood bags that avoid the use of phthalate plasticizers include blood bags made with (1,2- cyclohexane dicarboxylic acid diisononyl ester) (DINCH), (bis(2-ethylhexyl) adipate) (DEHA), and acetyl tributyl citrate (ATBC). Accordingly, in the broad context of the present invention, one may utilize the ZrO2nanoparticles and / or nanoporous zirconimum oxide macrostructure and / or cerium oxide nanoporous macrostructures herein, either directly in blood, or in the polymeric material to form the blood bag, or as a coating on a polymeric film insert, where such blood bags are made with one or more of DEHT, DINCH, DEHA and / or ATBC.

[0073] It can also be noted herein, that the ZrO2nanoparticles and / or nanoporous zirconimum oxide macrostructure and / or cerium oxide nanoporous macrostructures are contemplated to also provide an antimicrobial effect. For example, antimicrobial effect has been observed for the nanoporous cerium oxide macrostructure against two bacteria, S. epdermidis and E. coli. The minimum inhibitory concentration was observed to be 256 mg / L for both bacteria.

[0074] Working Examples

[0075] Films were produced herein via extrusion. In general, the plasticized PVC pellets, along with the selected additive (ZrCE nanoparticles or NCeONP) were mixed to a targeted loading level of the additive. Then, such mixture was extruded into films. For example, ZrO2nanoparticles at a 2.0 % (wt.) loading was prepared by mixing the ZrO2at such selected loading level with the PVC film (plasticized with DEHT) and then extruding such mixture into a film. In addition, the NCeONP macrostructures at a selected loading level (e.g., 5.0 % (wt.)) was combined with PVC pellets containing DEHT plasticizer, and then similarly extruded into a film.

[0076] Testing: The above referenced films containing 2.0 % (wt.) NCeONP macrostructrures or ZrO2nanoparticles were inserted into an existing commercial blood bag made using DEHP plasticizer. As illustrated in FIG. 14 a PVC film containing 2.0 % (wt.) NCeONP macrostructrures when inserted into a PVC blood bag made using DEHP plasticizer demonstrated a reduction in oxidative hemolysis, in RBC concentrates particularly at 6 weeks. Additionally, the presence of the NCeONP macrostructures or ZrO2nanoparticles at a 2.0 % (wt.) loading in the form of a film insert, positioned in the blood bag, were observed not to have an adverse impact on hemoglobin oxidation (%metHB) (FIG. 15), red cell distribution width (RDW-CV) versus storage week time (FIG. 16), glucose (mmol / L) versus storage week time (FIG. 17), lactate (mmol / L) versus storage week time (FIG. 18), percent oxygen saturation (measure of RBC capacity to bind oxygen) (FIG. 19). In FIGS. 14-19, error bars represent the standard error of the mean (Mean+SEM) of RBCs collected from 3 individuals (N=3). FIG. 20 illustrates the percent storage hemolysis of RBCs in a commercial blood bag versus storage week time, made with DEHP plasticized PVC film, containing a 2.0% (wt.) loading of NCeONP macrostructures in a DEHT PVC film insert.

[0077] Blood bags themselves were next evaluated for oxidative and storage hemolysis reduction of red blood cells. Specifically, PVC films using DEHT plasticizer were made using a 5.0 % (wt.) loading of the NCeONP macrostructures. The films were sealed using RF technology to produce the prototype blood bags. The RBCs in the bags was then tested. FIG. 21 shows the storage hemolysis of RBCs versus storage week time for a blood bag made of PVC plasticized with DEHT and containing 5.0 % (wt.) of NCeONP macro structures. Also shown is the European Union limit. FIG. 22 shows the percent oxidative hemolysis of RBCs versus storage week time for a blood bag made of PVC plasticized with DEHT and containing 5.0 % (wt.) of NCeONP macrostructures.

Claims

Claims:

1. A blood storage container comprising a blood storage surface comprising: zirconium dioxide ( ZrO2) nanoparticles and / or nanoporous zirconium dioxide nanoparticle macrostructures present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, and combinations thereof when such blood, blood product(s) and components thereof are stored in contact with the blood storage container surface.

2. The blood storage container of claim 1 wherein the ZrO2nanoparticles and / or nanoporous zirconium dioxide nanoparticle macrostructures are contained in a liquid composition present within the blood storage container and capable of mixing with blood, blood product, or components therefore stored within the blood storage container.

3. The blood storage container of claim 1 wherein the ZrO2nanoparticles and / or nanoporous zirconium dioxide nanoparticle macrostructures are contained on or in a polymeric film in said blood storage container wherein said blood, blood product(s) and components thereof are capable of contacting said ZrO2nanoparticles and / or zirconium oxide macrostructures.

4. The blood storage container of claim 1 wherein said container comprises polymeric material and said zirconium dioxide ( ZrO2) nanoparticles and / or nanonporous zirconium dioxide nanoparticle macrostructures are immobilized in said polymeric material and wherein said blood, blood product(s) and components thereof are capable of contacting said ZrO2nanoparticles and / or zirconium dioxide nanoparticle macrostructures.

5. The blood storage container of claim 1, wherein the blood storage container surface comprises a coating wherein the ZrO2nanoparticles and / or zirconium dioxide nanoparticle macrostructures form or are part of a composition that forms the coating on the blood storage container storage surface.

6. The blood storage container of claim 5 wherein said coating comprises a porous coating wherein said ZrO2nanoparticles and / or zirconium dioxide nanoparticle macrostructures are immobilized in said coating and wherein said blood, blood product(s) and components thereof are capable of contacting said ZrO2nanoparticles and / or zirconium dioxide nanoparticle macrostructures.

7. The blood storage container of claim 1 wherein the ZrO2nanoparticles have a diameter of 1.0 nm to 1000 nm.

8. The blood storage container of claim 1 wherein the zirconium oxide macro-structures have a macro-structure diameter in the range of 50 nm to 30,000 nm and macro- structure pore diameter in the range of 1 nm to 1000 nm.

9. The blood storage container of claim 1 wherein said container is made with a non- phthalate plasticizer.

10. A method of storing whole blood, blood product(s), components thereof or combinations thereof, comprising: a. supplying a blood storage container comprising a blood storage surface comprising zirconium dioxide ( ZrO2) nanoparticles and / or nanoporous zirconium dioxide nanoparticle macrostructures present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, or combinations thereof when such blood, blood product(s) andcomponents thereof are stored in contact with the blood storage container surface; b. storing blood, blood product(s), components thereof or combinations thereof in said blood storage container.

11. A method of storing blood, blood product products(s), components thereof or a combination thereof comprising: a. supplying blood, blood product(s) or components thereof; b. combining said blood, blood product(s), or components thereof with zirconium oxide nanoparticles and / or nanoporous zirconium oxide nanoparticle macrostructures wherein the concentration of said zirconium oxide nanoparticles and / or nanoporous zirconium oxide nanoparticle macrostructures is in the range of 1 pM to 1M.

12. A blood container insert having a surface, comprising a coating, wherein the coating comprises zirconium dioxide (ZrO2) nanoparticles and / or nanoporous zirconium dioxide nanoparticle macrostructures in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, or combinations thereof, when such blood, blood product(s) and components thereof are stored in contact with the insert inside of the blood storage container.

13. A blood storage container comprising a blood storage surface comprising: nanoporous cerium oxide nanoparticle (NCeONP) macrostructures present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, and combinations thereof when such blood, blood product(s) and components thereof are stored in contact with the blood storage container surface.

14. The blood storage container of claim 13 wherein the nanoporous cerium oxide nanoparticle (NCeONP) macrostructures are contained in a liquid composition present within the blood storage container and capable of mixing with blood, blood product or components therefore stored within the blood storage container.

15. The blood storage container of claim 13 wherein the nanoporous cerium oxide nanoparticle (NCeONP) macrostructures are contained on or in a polymeric film in said blood storage container wherein said blood, blood product(s) and components thereof are capable of contacting said nanoporous cerium oxide nanoparticle (NCeONP) macrostructures.

16. The blood storage container of claim 13, wherein the blood storage container surface comprises a coating wherein the nanoporous cerium oxide nanoparticle macrostructures form or are part of a composition that forms the coating on the blood storage container storage surface.

17. The blood storage container of claim 16 wherein said coating comprises a porous coating wherein said nanoporous cerium oxide nanoparticle macrostructures are immobilized in said coating and wherein said blood, blood product(s) and components thereof are capable of coming into contact with said nanoporous cerium oxide nanoparticle macrostructures.

18. The blood storage container of claim 13 wherein the nanoporous cerium oxide macro- structures have a macro-structure diameter in the range of 50 nm to 30,000 nm and macro-structure pore diameter in the range of 10 nm to 1000 nm.

19. The blood storage container of claim 13 wherein said container is made with a non- phthalate plasticizer.

20. A method of storing whole blood, blood product(s), components thereof or combinations thereof, comprising: a. supplying a blood storage container comprising a blood storage surface comprising nanoporous cerium oxide nanoparticle macrostructures present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, or combinations thereof when such blood, blood product(s) and components thereof are stored in contact with the blood storage container surface; b. storing blood, blood product(s), components thereof or combinations thereof in said blood storage container.

21. A method of storing blood, blood product products(s), components therefor or combination thereof comprising: a. supplying blood, blood product(s) or components thereof; b. combining said blood, blood product(s) or components thereof with nanoporous cerium oxide nanoparticle macrostructures wherein the concentration of said nanoporous cerium oxide nanoparticle macrostructures is in the range of 1 pM to 1M.

22. A blood container insert having a surface, comprising a coating, wherein the coating comprises nanoporous cerium oxide nanoparticle macrostructures in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, or combinations thereof, when such blood, blood product(s) and components thereof are stored in contact with the insert inside of the blood storage container.

23. A blood storage container comprising a blood storage surface comprising: zirconium dioxide (ZrO2) nanoparticles and cerium oxide nanoparticle macrostructures present in an amount effective to increase the useful storage life of blood, blood product(s), components thereof, and combinations thereof when such blood, blood product(s) and components thereof are stored in contact with the blood storage container surface.

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