Compositions and methods for tissue sterilization

Abstract

Systems, compositions, and methods of sterilization are presented in which a biological sample is sterilized using nitric oxide that is generated in a solution in which the sample is placed. In some embodiments, the nitric oxide is released from a nitric oxide precursor material that can be formed in situ from suitable reagents. Advantageously, sterilization can be effected at ambient temperature and pressure and uses nitric oxide as sterilant, thereby preserving biochemical and / or biomechanical properties of the sterilized tissue. Moreover, the sterilization process does not require dedicated equipment, and the sterilizing solutions can be discarded after use without adverse environmental impact.

Description

COMPOSITIONS AND METHODS FOR TISSUE STERILIZATION

[0001] This application claims priority to our co-pending U.S. Provisional Patent application with the serial number 63 / 447,811, which was filed 2 / 23 / 2023, and which is incorporated by reference herein.Field of the Invention

[0002] The field of the invention is sterilization of biological materials, especially as it relates to sterilization of biological donor tissue for storage and implantation.Background of the Invention

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0005] The use of human donor allografts for repair of damaged tissue has led to significant improvements in function and restoration of quality of life for many individuals receiving such allografts. Unfortunately, while the risk with tissue transplantation is relatively low, disease transmission nevertheless remains a substantial concern and therefore requires sterilization of allografts to reduce the chance of viral or bacterial transmission from a donor to a patient. There are numerous sterilization methods known in the art, however, many of these are unsuitable for allografts due to their harsh physical and / or chemical impact on the allograft. Indeed, and especially where the allograft is a tendon, maintaining the biomechanical properties of the tendon is critical to successful restoration of function.

[0006] Among other reported methods, tendons have been radiation sterilized with e-beam irradiation or gamma irradiation or sterilized with various chemicals such as supercriticalcarbon dioxide, ethylene oxide, hydrogen peroxide, chlorhexidine, or sterilized using multi- step processes (BioCleanse) using various chemical solutions and differing pressure cycles. Unfortunately, all of the currently known methods involve some level of damage or change to the tissue after processing, and in some cases will not sufficiently sterilize the tissue leaving the recipient at risk for disease transmission (see e.g., BMC Musculoskeletal Disorders (2021) 22:404). Consequently, sterilization of allografts often requires a compromise between sterility effectiveness and harshness of sterilization process, frequently impacting biochemical and biomechanical properties. In addition, many of the known processes will result in considerable quantities of waste materials that require treatment prior to disposal into the environment of sewage system.

[0007] In another example of tissue sterilization, nitric oxide radicals are released from a film placed into a subject’s mouth. The film is then illuminated to decompose nitroprusside in the film to so form the nitric oxide radical and to thereby topically treat microorganism promoted gingival disease as is described in US 2004 / 02652. While conceptually relatively simple, ingestion of the materials used may be problematic. Moreover, in situ generation of the nitric oxide required illumination, which is not always practicable or even desirable.

[0008] Thus, even though various compositions and methods of sterilization of biological tissues are known in the art, all or almost all of them suffer from several drawbacks. Therefore, there remains a need for improved compositions and methods that sterilize biological tissues in a gentle and effective manner that preserves biochemical and biomechanical properties, and that produce environmentally benign waste streams.Summary of The Invention

[0009] The inventive subject matter is directed to various compositions, systems, and methods of sterilization of various biological materials in which the biochemical and / or biomechanical properties of the biological materials are preserved, and that produce environmentally benign waste streams. The sterilization procedures presented herein can advantageously be performed at ambient temperatures and pressures without need for dedicated safety equipment.

[0010] In one aspect of the inventive subject matter, the inventor contemplates a method of sterilizing a biological tissue that includes a step of placing the tissue into a sterilizing solution for a time sufficient to reduce bacterial count in and / or on the tissue by at least 104-fold, wherein the solution contains a nitric oxide precursor material that decomposes to release nitric oxidein an amount sufficient to reduce the bacterial count, and wherein the nitric oxide precursor material is optionally generated in situ by reacting a first and a second reagent in the solution.

[0011] In preferred embodiments, the tissue is a human donor tissue for transplantation (e.g., a tendon, a cornea, bone, or skin), and / or the sterilizing solution is an aqueous solution. Where desired, the aqueous solution may further include an osmolarity adjusting agent, a nutrient, and / or a detergent. In further embodiments, the time for sterilization will be between 60 and 300 minutes, or between 300 and 1,200 minutes, or between 1,200 and 2,800 minutes (and even longer), and it is generally further preferred (but not necessary) that the tissue is sterilized in the solution at a temperature of between 4 °C and 37 °C and / or that the tissue is sterilized in the solution at a pressure of between 900 and 1,100 mbar. Most typically, contemplated methods will allow reduction of the bacterial count by at least 106-fold, or by at least 107-fold, and in some embodiments by at least 108-fold.

[0012] In exemplary embodiments it is contemplated that the tissue is a tendon, and that a mechanical tissue parameter of the tendon (e.g., failure load, ultimate tensile strength, and Young’s modulus of elasticity) after sterilization is substantially unchanged (e.g., changes no more than 10%).

[0013] In still further embodiments, the nitric oxide precursor material comprises a nitrosothiol, which may be added to the sterilizing solution prior to placing the tissue into the sterilizing solution, or the first and second reagents are a thiol containing compound and a nitrosating compound, respectively that react in the solution to form the nitrosothiol. Preferably, but not necessarily, wherein the nitrosothiol or wherein the thiol-containing compound and the nitrosating compound are provided in a dry formulation (e.g. , as a tablet or powder)

[0014] Therefore, the inventor contemplates a method of sterilizing a tendon tissue while maintaining a mechanical property of the tendon tissue (e.g., failure load, ultimate tensile strength, and / or Young’s modulus of elasticity). Such method will include a step of placing the tendon tissue into a sterilizing solution for a time sufficient to reduce bacterial count in and / or on the tissue by at least 104-fold, wherein the solution contains a nitric oxide precursor material that decomposes to release nitric oxide in an amount sufficient to reduce the bacterial count, and wherein the nitric oxide precursor material is optionally generated in situ by reacting a first and a second reagent in the solution.

[0015] It is generally preferred that in such methods the sterilizing solution is an aqueous solution, wherein the nitric oxide precursor material comprises a nitrosothiol, and wherein the first and second reagents are a thiol-containing compound and a nitrosating compound, respectively. Furthermore, it is preferred that the tissue is sterilized in the solution at a temperature of between 4 °C and 37 °C, and / or wherein the tissue is sterilized in the solution at a pressure of between 900 and 1,100 mbar. As will be appreciated, contemplated methods will be suitable to reduce the bacterial count by at least 106-fold.

[0016] Consequently, the inventor contemplates an intermediate sterilization composition that comprises a biological tissue that is submerged in a sterilizing solution. In such composition, the sterilizing solution contains a nitric oxide precursor material that decomposes in the solution to release nitric oxide in an amount sufficient to reduce bacterial count in and / or on the tissue. Most typically, the nitric oxide precursor material will have a chemical composition that releases nitric oxide in a quantity that reduces the bacterial count in and / or on the tissue by at least 104-fold after exposure of the tissue to the nitric oxide for equal or less than 6 hours.

[0017] In preferred embodiments, the mechanical property of the tissue remains substantially unchanged (e.g., failure load, ultimate tensile strength, and / or Young’s modulus of the tendon after sterilization changes no more than 10%), and the sterilizing solution has a temperature of between 4-37 °C and is at a pressure of between 900 and 1,100 mbar.

[0018] Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.Brief Description of The Drawing

[0019] FIG.l shows photographs of exemplary results for direct inoculation of a human tendon and subsequent sterilization according to the inventive subject matter along with a control.

[0020] FIG.2A and 2B show photographs of exemplary results for a human tendon preloaded with 24-hour bioburden and subsequent sterilization according to the inventive subject matter along with a control.

[0021] FIG.3 is a photograph of an experimental setup to determine mechanical properties of tendon materials tested with an exemplary tissue sterilizing solution.

[0022] FIG.4 is a graph depicting exemplary comparative stress strain curves of human tendon tested with an exemplary tissue sterilizing solution and sterile saline, respectively.

[0023] FIG.5 is a graph depicting exemplary comparative stress change curves over time for human tendon tested with an exemplary tissue sterilizing solution and sterile saline, respectively.Detailed Description

[0024] The inventor has discovered various compositions and methods of tissue sterilization that can be performed at ambient conditions (e.g., temperature between 20-25 °C, atmospheric pressure, and 20-80% relative humidity) using conventional reagents that generate nitric oxide in situ in an amount sufficient to sterilize the tissue upon contact within a relatively short period of time (e.g., between 2-6 hours, or 6 hours and 24 hours). Advantageously, the sterilization process takes place in a solution that can be modified to accommodate the specific needs of the tissue, and the solution can be replaced by a transport or storage solution after sterilization is complete.

[0025] Moreover, it should be noted that the solutions presented herein are able to sterilize a variety of biological tissues, including excised allografts, decellularized tissue, cellularized matrices, various soft tissues and hard tissue, membranes, and cells such as nerves, bone marrow cells, T-cells, etc. without damaging or changing biochemical and / or biomechanical properties of the tissue. In addition, it should be appreciated that the solutions presented herein are environmentally benign, do not require dedicated personal protective equipment for use, and can be discarded without further treatment.

[0026] In one embodiment a sterilizing solution comprises a nitrosothiol that decomposes at a rate sufficient to produce nitric oxide in an amount that is effective to generate a sterilizing solution. As will be readily appreciated, the nitrosothiol may be added to the solution (as a compound or in association with a carrier) or may be generated from precursor materials in situ. Thus, it should be appreciated that the nitric oxide is formed in the solution in situ from a precursor compound and that the nitric oxide exerts sterilizing action in the tissue while the tissue in in the solution. Advantageously, the solution is compatible with the particular tissue,and may therefore be adjusted to an appropriate osmolarity, nutrient and / or salt content, and may additionally include one or more pharmaceutically active agent to maintain or preserve a specific tissue state.

[0027] For example, the nitrosothiol may be S'-nitrosoglutathione or nitrosocysteine, which can be formed in a typically aqueous solution in situ by a reaction between glutathione or cysteine and sodium nitrite. Most typically, the nitrosothiol or other nitric oxide precursor will be present in the solution in an amount of between 1-20 mM, or between 20-50 mM, or between 50-100 mM, or even higher. As will be appreciated, the so prepared sterilizing solution may an isotonic solution (or may be adjusted to isotonicity using a suitable salt or other tonicity agent) and as such may be a 0.9% saline solution. The donor tissue is then placed into the sterilizing solution and incubated, typically at room temperature and atmospheric pressure for 2-6 hours, to achieve a desired degree of sterility (e.g., SAL of equal or less than 1 O'6). Upon sterilization and where desired, the donor tissue can be rinsed with sterile saline (or other suitable solution) and stored or shipped to its place of use.

[0028] With regard to suitable nitric oxide precursor materials, it should be appreciated that a wide variety of materials are deemed appropriate so long as such materials will generate nitic oxide in a sterilizing quantity over a predetermined period of time. Therefore, contemplated nitric oxide precursor materials will include reaction products from a thiol-containing compound and a nitrosating compound (which may be formed in situ in the sterilizing solution or which may have been previously prepared), reaction products from a thiolactone, a primary amine, and a nitrosating compound (which may be formed in situ in the sterilizing solution or which may have been previously prepared), reaction products from a thiol-containing alcohol, a silane, and a nitrosating compound (which may be formed in situ in the sterilizing solution or which may have been previously prepared), and / or a modified polymer that contains a nitric oxide precursor material. As will be appreciated, decomposition of the nitric oxide precursor materials will produce nitric oxide that then acts as the sterilant.

[0029] Moreover, in at least some embodiments, decomposition of the nitric oxide precursor material to generate nitric oxide can be controlled by control of specific reaction conditions (e.g., delivery of thermal or acoustic energy, illumination, or RF irradiation, change in pH, etc.). Additionally, it should be recognized that the type of nitric oxide precursor material may also determined at least in part the rate of decomposition. For example, nitric oxide precursor materials with a primary nitrosothiol group will decompose at a faster rate than those with asecondary nitrosothiol group. Therefore, nitric oxide release can be tuned to specific profiles using primary and / or secondary nitrosothiols, along with modification or adjustment of suitable environmental parameters. As will be readily appreciated, the nitric oxide precursor material may be provided as a final compound (typically in a dry formulation) that is then placed into a typically aqueous solution, or the nitric oxide precursor material may be formed in situ in the typically aqueous solution by a reaction of the components as indicated above.

[0030] For example, where the nitric oxide precursor material is formed in situ from a thiol- containing compound and a nitrosating compound, exemplary thiol-containing compounds include 1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione, dithioerythritol, 3,4- dimercaptotoluene, 2,3 -butanedithiol, 1,3-propanedithiol, 2-hydroxypropane thiol, 1- mercapto-2 -propanol, dithioerythritol and dithiothreitol. Other exemplary thiol-containing compounds include alpha-lipoic acid, methanethiol (CH3SH [m-mercaptan]), ethanethiol (C2H5SH [e-mercaptan]), 1 -propanethiol (C3H7SH [n-P mercaptan]), 2-propanethiol (CH3CH(SH)CH3 [2C3 mercaptan]), butanethiol (C4H9SH ([n-butyl mercaptan]), tert-butyl mercaptan (CXCThfSH [t-butyl mercaptan]), pentanethiols (C5H11SH [pentyl mercaptan]), coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothreitol, dithioerythritol, 2-mercaptoindole, transglutaminase, (1 l-mercaptoundecyl)hexa(ethylene glycol), (11 -mercaptoundecyl) tetra(ethylene glycol), (l l-mercaptoundecyl)tetra(ethylene glycol) functionalized gold nanoparticles, 1,1', 4', 1 "-terphenyl -4-thiol, 1,11 -undecanedithiol, 1,16-hexadecanedithiol, 1 ,2-ethanedithiol, 1,3-propanedithiol, 1,4-benzenedimethanethiol, 1 ,4-butanedithiol, 1 ,4-butanedithiol diacetate, 1,5-pentanedithiol, 1,6-hexanedithiol, 1,8- octanedithiol, 1 ,9-nonanedithiol, adamantanethiol, 1 -butanethiol, 1 -decanethiol, 1- dodecanethiol, 1 -heptanethiol, 1 -heptanethiol purum, 1 -hexadecanethiol, 1 -hexanethiol, 1- mercapto-(triethylene glycol), l-mercapto-(triethylene glycol) methyl ether functionalized gold nanoparticles, 1 -mercapto-2-propanol, 1 -nonanethiol, 1 -octadecanethiol, 1 -octanethiol, 1- octanethiol, 1 -pentadecanethiol, 1 -pentanethiol, 1 -propanethiol, 1 -tetradecanethiol, 1- tetradecanethiol purum, 1 -undecanethiol, l l-(lH-pyrrol-l-yl)undecane-l -thiol, 11 -amino- 1- undecanethiol hydrochloride, 11 -bromo- 1 -undecanethiol, 11 -mercapto- 1 -undecanol, 11- mercapto-1 -undecanol, 11-mercaptoundecanoic acid, 11-mercaptoundecanoic acid, 11- mercaptoundecyl trifluoroacetate, 11 -mercaptoundecylphosphoric acid, 12- mercaptododecanoic acid, 12-mercaptododecanoic acid, 15 -mercaptopentadecanoic acid, 16- mercaptohexadecanoic acid, 16-mercaptohexadecanoic acid, 1H,1H,2H,2H- perfluorodecanethiol, 2,2'-(ethylenedioxy)diethanethiol, 2,3-butanedithiol, 2-butanethiol, 2-ethylhexanethiol, 2 -methyl- 1 -propanethiol, 2-methyl-2 -propanethiol, 2-phenylethanethiol, 3,3,4,4,5,5,6,6,6-nonafluoro-l-hexanethiol purum, 3 -(dimethoxymethylsilyl)- 1 -propanethiol, 3 -chloro- 1 -propanethiol, 3 -mercapto- 1 -propanol, 3-mercapto-2-butanol, 3-mercapto-N- nonylpropionamide, 3 -mercaptopropionic acid, 3-mercaptopropyl-functionalized silica gel, 3- methyl-1 -butanethiol, 4,4'-bis(mercaptomethyl)biphenyl, 4,4'-dimercaptostilbene, 4-(6- mercaptohexyloxy)benzyl alcohol, 4-cyano-l -butanethiol, 4-mercapto-l -butanol, 6- (ferrocenyl)hexanethiol, 6-mercapto-l -hexanol, 6-mercaptohexanoic acid, 8 -mercapto- 1- octanol, 8-mercaptooctanoic acid, 9-mercapto-l -nonanol, biphenyl-4, 4 '-dithiol, butyl 3- mercaptopropionate, copper(I) 1 -butanethiolate, cyclohexanethiol, cyclopentanethiol, decanethiol functionalized silver nanoparticles, dodecanethiol functionalized gold nanoparticles, dodecanethiol functionalized silver nanoparticles, hexa(ethylene glycoljmono- l l-(acetylthio)undecyl ether, mercaptosuccinic acid, methyl 3 -mercaptopropionate, octanethiol functionalized gold nanoparticles, PEG dithiol, 5-(l l-bromoundecyl)thioacetate, 5'-(4-cyanobutyl)thioacctatc, thiophenol, triethylene glycol mono- 11 -mercaptoundecyl ether, trimethylolpropane tris(3-mercaptopropionate), [1 l-(methylcarbonylthio)undecyl]tetra (ethylene glycol), m-carborane-9-thiol, p-terphenyl-4,4"-dithiol, tert-dodecylmercaptan, and / or or tert-nonyl mercaptan.

[0031] In certain embodiments, the thiol-containing compound includes a cysteine or derivative thereof, a thiol-derivatized polymer or fdler, or a combination thereof. In embodiments when the cysteine or derivative thereof is utilized, the cysteine or derivative thereof may comprise cysteine, glutathione, acetyl cysteine, penicillamine, acetylpenicillamine, '-nitroso-n-acetylpenicillamine, bucillamine, or combinations thereof. It is to be appreciated that the thiol-containing compound may be included as part of a peptide or other macromolecules so long as the thiol-containing compound is compatible with the components of the multi-component composition. In embodiments when the cysteine or derivative thereof is utilized as part of a peptide, the peptide may include any combination of amino acids so long as the peptide includes the cysteine or derivative thereof as at least one of the constituents of the peptide. Non-limiting examples of suitable cysteines or derivatives thereof are described in a journal article titled ‘\S'-Nitrosothiol Detection via Amperometric Nitric Oxide Sensor with Surface Modified Hydrogel Layer Containing Immobilized Organoselenium Catalyst” cited as Langmuir 2006, 22, 25, 10830-10836, which is incorporated by reference in its entirety. Further considerations, aspects, and embodiments aredescribed in our copending International patent application WO 2023 / 219811, which is incorporated by reference herein.

[0032] In another example, where the nitric oxide precursor material is formed from a thiolactone, a primary amine, and a nitrosating compound, especially contemplated thiolactones include those with an a-acetothiolactone group, a P-propiothiolactone group, a - butyrothiolactone group, a 8-valerothiolactone group, a c-caprothiolactone group, a C,- enanthothiolactone group, a r|-caprylothiolactone group, and a 0-pelargothiolactone group. In some embodiments, the thiolactone is an amine-containing thiolactone, such as a thietanone (e.g., N-(2,2-Dimethyl-4-oxo-3-thietanyl)acetamide).

[0033] In various embodiments, suitable primary amines in conjunction with the teachings presented herein include cysteine or derivatives thereof, lysine or derivatives thereof, butylamine or derivatives thereof, or a combination thereof. In these and other embodiments, the primary amine contains a thiol functional group. In exemplary embodiments, the cysteine or derivative thereof comprises cysteine, glutathione, acetyl cysteine, penicillamine, acetylpenicillamine, S'-nitroso-n-acetylpenicillamine, bucillamine, or combinations thereof. As will be readily appreciated, that thiol functional group may also participate in a nitrosation reaction and as such provide an additional nitrosyl group for decomposition to nitric oxide. Further considerations, aspects, and embodiments are described in our copending International patent application with the serial number PCT / US23 / 80533, which was fded 11 / 20 / 2023, and which is incorporated by reference herein. As will be readily appreciated, secondary and tertiary amine compounds are also expressly contemplated for use herein.

[0034] In still further examples, where the nitric oxide precursor material is formed from a thiol-containing alcohol, a silane, and a nitrosating compound, the thiol-containing alcohol may be 3 -mercapto-3 -methyl- 1 -butanol, 2-mercaptoethanol, 3 -mercaptohexanol, 4-mercapto-3- methyl-2-butanol, 3-mercapto-2-methyl-l -butanol, 3 -mercapto-2-methyl-l -pentanol, (+ / - )-4- mercapto-4-methyl-2-pentanol, 2-mercapto-2-methyl- 1 -pentanol, 4-mercapto-4-methyl- pentan-2-ol, 3 -mercapto hexan-l-ol, or combinations thereof, and the silane may be tetramethoxysilane, tetraethoxysilane. Further considerations, aspects, and embodiments are described in our copending International patent application with the serial number PCT / US23 / 79150, which was fded 11 / 08 / 2023, and which is incorporated by reference herein

[0035] Contemplated nitrosating compounds suitable for use with the above reagents include those that can act as a nitroso group donor and will therefore be a compound of formula NOX where X is an organic or inorganic anion or a group OR2 where R2 is an organic group. In such compounds, X may be an organic anion derived from a carboxylic acid, e.g., an alkane carboxylic acid containing 2 - 7 carbon atoms (e.g., nitrosating agents of this type include acetyl nitrite and propionyl nitrite). Where X is an inorganic anion, X may be derived from, for example, a mineral acid, e.g., a halide ion such as chloride or bromide or a sulphate ion, or from a Lewis acid, e.g., a boro fluoride ion. Other inorganic anions include hydroxide and sulphonate. Nitrosating compounds of this type thus include nitrosyl chloride, nitrosyl sulphate, nitrosyl borofluoride, nitrous acid and Fremys salt (potassium nitrosyldisulphonate). Where X is a group of formula OR2 the organic group R2 may be, for example, a lower alkyl group, such as containing 1 - 9 carbon atoms, e.g., ethyl, n-propyl, isopropyl, n-butyl, t-butyl or isopentyl.

[0036] In certain embodiments, the nitrosating compound comprises a nitrite. The nitrite may comprise sodium nitrite, calcium nitrite, potassium nitrite, tetrabutylammonium nitrite, dicyclohexylammonium nitrite, butylnitrite, isobutylnitrite, t-butylnitrite, amylnitrite, pentylnitrite, nitrite salts, ion paired nitrite, silver nitrite, zinc nitrite, iron nitrite, copper nitrite, transition metal-nitrite compounds, or combinations thereof. In still further embodiments, nitric oxide gas may be used as a nitrosating agent.

[0037] In certain embodiments, the reaction of the nitrosating compound(s) to produce the nitric oxide precursor material will typically be performed in an aqueous solvent, such as water or a mixture of water and methanol or ethanol. However, and depending on the type of reactants, the solvent may also comprise an organic solvent, such as tetrahydrofuran. Other non-limiting examples of suitable solvents include various aromatics, aliphatics, ketones, such as methyl ethyl ketone, isobutyl ketone, ethyl amyl ketone, acetone, alcohols, such as methanol, ethanol n-butanol isopropanol esters, such as ethyl acetate, glycols, such as ethylene glycol propylene glycol ethers, such as tetrahydro furan, ethylene glycol mono butyl ether, or combinations thereof. Therefore, it should be recognized that where the nitric oxide precursor material is prepared in situ in the solution for sterilization, solvents or solvent mixtures that are physiologically acceptable (i.e., will not denature tissue or cells, and / or will not change mechanical properties of the tissue) are especially preferred, whereas no limitations are present where the nitric oxide precursor material is produced before combining the nitric oxide precursor material with the solution for sterilization.

[0038] In yet another example, where the nitric oxide precursor material is a polymer that is modified to contain a nitric oxide precursor, the polymer may be a carrier that retains at least temporarily contemplated nitric oxide precursor material or reagents used to form the nitric oxide precursor material, or the polymer may be chemically modified to include covalently bound nitric oxide precursor material. As will be readily appreciated, such polymers may be soluble or insoluble, swellable or non-swellable, and / or may be organic polymers or inorganic polymers. Therefore, it should be appreciated that all precursor materials and reagents that form the precursor materials can be blended into polymers, solution phases, or powders, and used to generate nitric oxide for creating a wide array of NO donating / generating entities by blending the nitric oxide precursor into the matrix (polymer or powder or embedded in and on solid carriers) or for creating solutions that generate NO in a controlled and predicable manner from the matrix / solution phase. Exemplary suitable polymeric materials and their production are described in our copending International patent application WO 2023 / 205125, which is incorporated by reference herein.

[0039] With respect to suitable quantities for nitric oxide release it is generally contemplated that the nitric oxide precursor material will be present in the solution in an amount of at least 0.1 mM, or at least 0.5 mM, or at least 1.0 mM, or at least 3.0 mM, or at least 6.0 mM, or at least 10 mM, or at least 25 mM, or at least 50 mM, or at least 100 mM, or even more to release nitric oxide in sterilizing quantities over time (e.g., at least 5 min, or at least 15 min, or at least 30 min, or at least 45 min, or at least 60 min, or at least 90 min, or at least 120 min, or at least 3 hours, or at least 4 hours, or at least 6 hours, or even longer). Therefore, suitable quantities of nitric oxide precursor materials will be in the range of 0.1-1.0 mM, or 1.0-10 mM, or 10-25 mM, or 25-50 mM, or 50-100 mM. As will be readily recognized, where reagents are added to a solution to so produce the nitric oxide, the quantities of the respective agents will be chosen such as to yield contemplated nitric oxide precursor materials in amounts as discussed above.

[0040] Therefore, it should be appreciated that the sterilizing solutions can be produced at the time of use by combining solid or liquid reactants before adding the reactants to the sterilizing solution, or by adding the solid or liquid reactants to the sterilizing solution. Similarly, the sterilizing solutions can also be produced at the time of use by combining a nitric oxide precursor material with the solution. In still other embodiments, the sterilizing solution already contains nitric oxide precursor materials or reagents to produce the nitric oxide precursor materials, and the nitric oxide release is started by irradiation, change in pH, illumination, etc.Viewed from a different perspective, the nitric oxide in contemplated solutions will preferably be formed in situ in the solution at the time of use. As noted above, suitable solutions will typically be aqueous solutions, but non-aqueous solutions are not excluded herein. Moreover, the sterilizing solution can include additional ingredients that may provide a specific function (e.g., adjust osmolarity and / or pH, add nutrients, antifungal activity, protease inhibition, add detergents, etc.).

[0041] As will be readily appreciated, the nitric oxide precursor material or the components for in situ generation of the nitric oxide precursor material can be provided in a variety of formats, and especially suitable formats include liquids where the components are typically physically separate liquids that can then be combined (per se or in a solution) prior to use. On the other hand, where the nitric oxide precursor material is a solid (or coupled to a solid), it is contemplated that such solid may be formulated as a tablet or a powder. Likewise, where the components for in situ generation of the nitric oxide precursor material are solids, the solids may be provided separately, and more preferably, in a combination in dry form (e.g. , as a tablet or powder). Adding the solid form into a solution will then liberate the nitric oxide precursor or allow reaction of the components to so form the nitric oxide precursor material.

[0042] Regardless of the type of formulation and reagent used, it is contemplated that the sterilizing solution is prepared within a relatively short time of use (i.e., contact with the cells or tissue). For example, the time between preparation and use of the sterilizing solution will typically be less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, or less than 5 minutes. Likewise, it is generally preferred that the period for contact of the cells or tissue with the sterilizing solution will be between 15 seconds to 1 minute, or between 1 and 15 minutes, or between 15 and 60 minutes, or between 60 minutes and 180 minutes or between 2 hours and 6 hours, and in some cases even longer (e.g., where the solution is also used for temporary storage for between 12 hours and three days). However, significantly longer times are also expressly contemplated herein and include times between 6 and 12 hours, or between 12-18 hours, or between 18 and 24 hours, or between 24 and 48 hours. Thus, sterilization can be performed as a relatively fast process without subsequent storage or as a slow process that may include additional storage time in the same solution without further sterilization (e.g. , not needed or no more nitric oxide precursor material available).

[0043] Therefore, suitable time frames for sterilizing the cells or tissues will be at least 1 minute, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, and typically less than 48 hours, less than 24 hours, less than 12 hours, less than 10 hours, less than 8 hours, or even less than 6 hours. Such time frames will generally be suitable to achieve sterility to reduce bacterial count in and / or on the tissue by at least 104-fold, or at least 105-fold, or at least 106-fold, or at least 107-fold, or at least 108-fold. Viewed from a different perspective, contemplated sterilizing solutions will be able to produce (typically within the above time frame) an SAL of equal or less than 10-4, or an SAL of equal or less than 10'5, or an SAL of equal or less than 10'6.

[0044] In view of the present disclosure, it should be recognized that the type of biological materials for sterilization may vary considerably and will include tissue samples for transplantation or investigation (e.g., comeal tissues, skin, bone, bone marrow, etc.), cellularized and decellularized (synthetic and natural) matrices, and even organs for transplantation. Most typically, such tissue samples will be mammalian tissues, and most typically human tissues (e.g. , for autologous or heterologous transplantation). Alternatively, it should also be appreciated that the tissues for sterilization contemplated herein will also include allografts and even xenografts, and suitable donor tissues for xenografts will include bovine tissues, porcine tissues, lizard tissues, fish tissues, etc., and indeed any tissue that is suitable for a xenotransplant from one species to another. Additionally, it should be recognized that the sterilizing solutions presented herein may also be used to decontaminate biological materials (cells, tissues, cell-containing or cell-free bodily fluids, aspirates, exudates, etc.) prior to disposal.

[0045] Among other benefits of using contemplated sterilizing solutions, it is particularly noted that cell and tissues sterilized by such solutions will remain substantially unchanged with respect to their biochemical and / or mechanical properties. Such is particularly advantageous where the cells or tissues are donor cells or donor tissues to be implanted or transplanted. As used herein, the term “substantially unchanged” means that a biochemical and / or mechanical property will change less than 10% in its property. For example, the tensile strength and / or modulus of tendon material will not change more than 10% absolute form before to after sterilization. Similarly, optical transparency of comeal tissue will not change more than 10% absolute form before to after sterilization. Similarly, skin elasticity will not change more than 10% absolute form before to after sterilization. In still further contemplated aspects, thesterilizing solutions presented herein may also be employed as a wound wash or rinse fluid for bodily surfaces (or other fomites) that are suspected to carry a microbial contamination. Once more, generation of the sterilizing fluid at the point of care / use is particularly advantageous, especially in field or emergent medical use.Examples

[0046] While the following examples provide specific guidance for the preparation and use of the sterilizing solutions presented herein, it should be appreciated that the skilled artisan can modify the examples using the teachings above to arrive at further sterilizing solutions suitable for use with biological materials.

[0047] Exemplary Sterility Test

[0048] In one example, dry reagent powder was added to tissue bathing solutions such that NO was generated in situ at a level sufficient to sterilize human tendon tissue. In the instant examples, the inventor tested solutions containing 50-100 mM .S'-nitrosoglutathionc or nitrosocysteine that were formed in situ by combining glutathione (or cysteine) and sodium nitrite in 0.9% saline, phosphate buffered saline (PBS), and Allowash (commercially available from LifeNet Health, Virginia Beach, VA 23453). These solutions were able to sterilize human tendon tissue that was directly inoculated with E.coli as described in more detail below. After the tissue was sterilized, the sterilizing solution was treated with a mild base to decompose the remaining NO-donors and subsequently neutralized with a mild acid, resulting in a salt solution that was disposed without need for chemical disposal requirements.

[0049] Pieces of human tendon (approximately 2 cm) were soaked in sterile PBS for 20 hours to remove residual antibiotics. The tendon pieces were then injected with 100 ml of 7.4 x 106CFU of E.coli and placed in 2 mL of tryptic soy broth (TSB). To the control tendons, 2 mL of sterile water were added, to the treatment tendons, 2 mL of the sterilizing solution (50-100 mM '-nitrosoglutathione or nitrosocysteine in 0.9% saline, phosphate buffered saline (PBS), or Allowash) were added and gently shaken on an orbital shaker. The tendons were allowed to incubate, and samples of the bathing solutions were removed at 0.5, 1, 2, 3, and 4 hours. Aliquots of these samples were plated out on Muller Hinton agar plates and incubated at 35 °C for 48 hours. Notably, as can be seen from FIG.l, after only 2 hours incubation of the tendon, no bacteria remained in the tendon solution treated with ‘Sterile Solutions’, which corresponds to a log6 reduction in bacterial load.

[0050] After 24 hours, the control tendon was placed in a fresh 2 mL of TSB and treated with fresh 2 mL of the sterilizing solution. The E.coli load was estimated to be 108CFUs. The tendon was allowed to incubate with gentle shaking and samples were removed at 0, 2, 4, 6, 22 hours. Aliquots of these samples were plated out on Muller Hinton agar plates and incubated for at 35°C for 48 hours. Notably, as can be seen from FIG.2, after 6 hours, all bacteria in the tendon were eliminated, which corresponds to a log8 reduction in bacterial load.

[0051] Exemplary mechanical Property Test

[0052] In another example, a tissue sterilizing solution was prepared by dissolving 1 g of reduced glutathione and 0.25 g of sodium nitrite in 250 mL of DI water (equivalent to 4 mg / mL reduced glutathione and 1 mg / mL sodium nitrite). The solution was inverted / gently agitated until all solids were completely dissolved and used within 2 hours of preparation. The so prepared solution turned from an initial pink color to a red color within about 5 minutes.

[0053] Human tendon was obtained from CTS. Tendon was dissected from the longitudinal midline. One half was subjected to the tissue sterilizing solution while the other half was used as a control soaked in sterile saline solution.

[0054] Human tendon was transferred into a 50 mL centrifuge tube. To completely submerge the tissues, 30 mL tissue sterilizing solution was added into the centrifuge tube. Then the tube was capped and tightened. Likewise, the control tissues was soaked in 30 mL sterile saline. Both tubes were placed into the 35 °C incubator for a 24-hour incubation.

[0055] After treatment, all tissues were rinsed with sterile PBS (phosphate buffered saline). Tissues were cut into about 3 -inch-long specimens for tensile testing. The dimension of every specimen was summarized in Table 1 in which L denotes major axes of the tissue cross-section (two positions, LI, L2, were chosen), W denotes minor axes of the tissue cross-section (two positions, Wl, W2, were chosen). Samples 1-4 were treated with tissue sterilizing solution, Controls Ctrl 1-4 were treated with sterile saline solution.Table 1

[0056] Standard construction material testing (CMT) equipment was used for the mechanical tests. The parameters used for the tensile test are as follows: Sample length (lo) = 20 mm; Cycle number = 30; Frequency = 1.5 Hz; Cycle start length = 2 mm; Cycle end length = 4 mm. The following parameters were recorded: Time (t, second), Displacement (Al, mm), and Force (F, N). Stress was calculated as Force / Area, and Strain was calculated as difference in distance between the two sample clips at time zero and the two sample clips at conclusion / distance between the two sample clips at time zero. The Secant modulus was estimated by calculating the slope of a line drawn based on the stress-strain diagram (from strain 14% to 19%). FIG.3 is a photograph of the experimental setup.

[0057] FIG.4 is a graph depicting exemplary comparative stress strain curves, and Table 2 shows the maximum stress and moduli of the human tendon samples.

[0058] A T-test was used to determine whether there was a significant difference between the tissue sterilizing solution treated group and the control group. Here, the p-values were 0.248049 and 0.149467, respectively for Secant modulus and maximum stress, indicating no statistically significant difference at / ?<() .05. Stress changes over time were measured and FIG.5 depicts an exemplary comparative graph illustrating stress changes over time during 30 cycles of the tensile test.

[0059] Notably, as can be seen from the data above, the tissue sterilizing solutions contemplated herein effectively sterilized human tissue without altering mechanical properties in a statistically significant manner. Indeed, it should be appreciated that the tissue sterilizing solutions contemplated herein will not require high temperature, high pressure, strong acidity, strong base, or corrosive components, which are commonly needed, and which significantly alter mechanical properties of human tissue under conditions that ensure sterility.

[0060] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” As used herein, the terms "about" and "approximately", when referring to a specified, measurable value (such as a parameter, an amount, a temporal duration, and the like), is meant to encompass the specified value and variations of and from the specified value, such as variations of + / -10% or less, alternatively + / -5% or less, alternatively + / -1% or less, alternatively + / -0.1% or less of and from the specified value, insofar as such variations are appropriate to perform in the disclosed embodiments. Thus, the value to which the modifier "about" or "approximately" refers is itself also specifically disclosed. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Unless noted otherwise or context dictates, all percentages indicated are percent by weight.

[0061] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0062] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. As also used herein, and unless the context dictates otherwise, the term "coupled to" is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements). Therefore, the terms "coupled to" and "coupled with" are used synonymously.

[0063] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exchisive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification or claims refer to at least one of something selected from the group consisting of A, B, C .... and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

CLAIMSWhat is claimed is:

1. A method of sterilizing a biological tissue, comprising: placing the tissue into a sterilizing solution for a time sufficient to reduce bacterial count in and / or on the tissue by at least 104-fold; wherein the tissue is a donor tissue for transplantation; wherein the sterilizing solution contains a nitric oxide precursor material that decomposes in situ to release nitric oxide in an amount sufficient to reduce the bacterial count; and wherein the nitric oxide precursor material is optionally generated in situ by reacting a first and a second reagent in the solution.

2. The method of claim 1, wherein the tissue is a human donor tissue.

3. The method of claim 2, wherein the tissue is a tendon, a cornea, bone, or skin.

4. The method of claim 1, wherein the sterilizing solution is an aqueous solution.

5. The method of claim 4, wherein the aqueous solution further comprises at least one of an osmolarity adjusting agent, a nutrient, and a detergent.

6. The method of claim 1, wherein the time is between 60 and 2800 minutes.

7. The method of claim 1, wherein the tissue is sterilized in the solution at a temperature of between 4 °C and 37 °C, and / or wherein the tissue is sterilized in the solution at a pressure of between 900 and 1,100 mbar.

8. The method of claim 1, wherein the bacterial count is reduced by at least 106-fold.

9. The method of claim 1, wherein the bacterial count is reduced by at least 108-fold.

10. The method of claim 1, wherein the tissue is a tendon, wherein a tissue parameter of the tendon after sterilization changes no more than 10%, and wherein the tissue parameter is selected from the group consisting of failure load, ultimate tensile strength, and Young’s modulus of elasticity.

11. The method of claim 1, wherein the nitric oxide precursor material comprises a nitrosothiol.

12. The method of claim 11, wherein the nitrosothiol is added to the sterilizing solution prior to placing the tissue into the sterilizing solution.

13. The method of claim 1, wherein the nitric oxide precursor material comprises a nitrosothiol, and wherein the first and second reagents are a thiol-containing compound and a nitrosating compound, respectively.

14. The method of claim 13, wherein the nitrosothiol or wherein the thiol-containing compound and the nitrosating compound are provided in a dry formulation that upon addition to a solution forms the sterilizing solution.

15. A method of sterilizing a tendon tissue while maintaining a mechanical property of the tendon tissue, comprising: placing the tendon tissue into a sterilizing solution for a time sufficient to reduce bacterial count in and / or on the tissue by at least 104-fold; wherein the solution contains a nitric oxide precursor material that decomposes in situ to release nitric oxide in an amount sufficient to reduce the bacterial count; and wherein the nitric oxide precursor material is optionally generated in situ by reacting a first and a second reagent in the solution.

16. The method of claim 15, wherein the sterilizing solution is an aqueous solution, wherein the nitric oxide precursor material comprises a nitrosothiol, and wherein the first and second reagents are a thiol-containing compound and a nitrosating compound, respectively.

17. The method of claim 16, wherein the nitrosothiol or wherein the thiol-containing compound and the nitrosating compound are provided in a dry formulation that upon addition to a solution forms the sterilizing solution.

18. The method of claim 17, wherein the dry formulation is a tablet or a powder.

19. The method of claim 15, wherein the tissue is sterilized in the solution at a temperature of between 4 °C and 37 °C, and / or wherein the tissue is sterilized in the solution at a pressure of between 900 and 1,100 mbar.

20. The method of claim 15, wherein the bacterial count is reduced by at least 106-fold.

21. The method of claim 15, wherein the mechanical property is selected form the group consisting of failure load, ultimate tensile strength, and / or Young’s modulus of elasticity.

22. An intermediate sterilization composition, comprising: a biological tissue submerged in a sterilizing solution that contains a nitric oxide precursor material that decomposes in the solution to release nitric oxide in an amount sufficient to reduce bacterial count in and / or on the tissue; wherein the nitric oxide precursor material has a composition that releases nitric oxide in a quantity that reduces the bacterial count in and / or on the tissue by at least 104-fold after exposure of the tissue to the nitric oxide for equal or less than 6 hours.

23. The sterilization composition of claim 22, wherein the nitric oxide precursor material is a nitrosothiol.

24. The sterilization composition of claim 22, wherein the biological tissue is sterilized in the solution at a temperature of between 4 °C and 37 °C, and / or wherein the tissue is sterilized in the solution at a pressure of between 900 and 1,100 mbar.

25. The sterilization composition of claim 22, wherein a mechanical property of the biological tissue changes no more than 10% between before and after sterilization.

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