Exosome-coated oxygen nanobubble-laden hydrogel

Abstract

A novel strategy for wound healing involving the coating of oxygen nanobubbles with exosomes and incorporating them into a polyvinyl alcohol (PVA) / gelatin hybrid hydrogel. In this work, we not only mitigate wound hypoxia but importantly have an efficient delivery method of exosomal nanoparticles, which to date has been confounded by very low to lack of uptake by cells in hypoxia. Furthermore, the self-healing properties of the hydrogel, along with its gelatin component, aids in hemostasis, thereby benefiting acute and surgical wound healing. Additionally, the crosslinking bonds within the hydrogel facilitate the decomposition of hydrogen peroxide (H2O2), alleviating wound inflammation. The multifunctional hydrogel provides for enhanced wound healing through increased angiogenesis, enhanced exosome delivery, hypoxia mitigation, and inflammation inhibition.

Description

[0001] EXOSOME-COATED OXYGEN NANOBUBBLE-LADEN HYDROGEL

[0002] RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 631,255 filed April 8, 2024, which is incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] Poor wound healing following trauma and surgical procedures constitutes a pressing global medical issue, impacting millions of individuals annually and posing substantial challenges to healthcare professionals. This concern arises due to the adverse effects on patients' quality of life, heightened psychosocial stress, and the considerable financial burden associated with prolonged clinical wound management. Statistics reveal that inadequate wound healing affects 6.5 million patients in the US, and among all wound types, surgical wounds - being the costliest - significantly contribute to total Medicare spending. The main goals in managing wounds encompass expeditious wound closure and the attainment of a scar (or lack thereof) that is both functional and aesthetically pleasing. However, anomalous wound healing can be triggered by the deviance of inflammation and hypoxia from their typical patterns during the normal healing process, thereby inducing delayed wound closure, keloid formation, and the development of hypertrophic scars.

[0006] Adipose-derived stem cell (ADSC)-derived exosomes have emerged as promising therapeutic agents in tissue regeneration. This is primarily attributed to their capability to function as intercellular communicators and harbor a rich cargo of bioactive molecules, including proteins, nucleic acids, and lipids. These molecules contribute to wound healing by exerting anti-inflammatory effects, inhibiting apoptosis, promoting angiogenesis, and facilitating enhanced cell migration and proliferation. Recent studies emphasize the potential of exosome-loaded hydrogels as multifunctional dressings for both acute and chronic wound healing. The data showcases their capability to mitigate oxidative stress, stimulate angiogenesis, and enhance fibroblast migration, contributing positively to all phases of the wound healing process. Notably, exosomes offer several advantages over stem cells, as they are considered safer, lack tumorigenic potential, and pose a minimal risk of embolism. Nevertheless, recent investigations have revealed that the intracellular cargo delivery efficiency of exosomes is compromised under wound hypoxic conditions due to activated hypoxia-induced endocytic recycling. This limitation significantly impedes their therapeutic efficacy and necessitates innovative approaches to enhance exosome delivery in hypoxic wound tissues.

[0007] The effective management of hypoxia and inflammation is of paramount importance for treating wounds. Accordingly, the development of next generation advanced wound dressings is greatly needed. SUMMARY

[0008] This disclosure provides a wound healing composition that also addresses wound inflammation and hypoxia utilizing ADSC-derived Exosome coated BSA-based Oxygen nanobubbles (EBO) embedded within a self-healing hydrogel matrix as a multifunctional wound dressing. The oxygen supply component, oxygen nanobubbles (ONB), is formed by encapsulating nanoscale oxygen bubbles within a glycosylated protein conjugate composed of dextran-conjugated bovine serum albumin (BSA).

[0009] Accordingly, this disclosure provides an oxygen releasing hydrogel comprising: a) exosome coated oxygen nanobubbles, wherein the exosome coated oxygen nanobubble comprises: i) an oxygen gas nanobubble encapsulated in a glycosylated protein-conjugate shell; and ii) an outer shell comprising isolated exosomes, wherein the isolated exosomes encapsulate the glycosylated protein-conjugate shell; and b) a hybrid hydrogel comprising an ionic-crosslinked polymer, a heteroatom substituted polymer, and a crosslinker that crosslinks the heteroatom substituted polymer; wherein the exosome coated oxygen nanobubbles are substantially evenly distributed in the hybrid hydrogel to form the oxygen releasing hydrogel.

[0010] This disclosure also provides a method for healing and / or for enhancing the healing of a wound comprising: a) contacting a wound and an effective amount of an oxygen releasing hydrogel described herein to the wound; b) covering the treated wound and the oxygen releasing hydrogel with a dressing for about 1 day to about 3 days; c) cleaning the treated wound; and d) optionally repeating steps a) to c); to thereby enhance and / or heal the wound.

[0011] The invention provides for the use of the compositions described herein for use in medical therapy. The medical therapy can be treating a wound, for example, a laceration from an injury or surgery. The invention also provides for the use of a composition as described herein for the manufacture of a dressing or bandage to treat a wound in a mammal, for example, a laceration in a human.

[0012] The invention provides therapeutic methods of treating a wound in a mammal, which involves administering to a mammal having a wound an effective amount of a composition described herein. A mammal includes a primate, human, rodent, canine, feline, bovine, ovine, equine, swine, caprine, bovine and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

[0014] Figure 1A-F. Synthesis and characterization of EBO (see Figure 20). Diameter distribution (A) and zeta potential values (B) of ONB and EBO (n=3). (C) TEM images of (i) exosomes; (ii) ONB; (iii) EBO. Scale bar = 50 nm. (D) The browning intensity of early (A294) and late (A420) MRP (n=3). (E) Infrared spectra of BSA and ONB. (F) SDS-PAGE of different formulations. Lanes: 1. Natural BSA; 2. Mixture of BSA and dextran sulfate; 3. Ultrasonicated BSA; 4. Shell; 5. ONB; 6. Exosomes; 7. EBO. Data are shown as mean ± standard deviation (SD).

[0015] Figure 2. Characterizations of EBO-Gel. (a) Formation of EBO-Gel. (b) SEM images of Blank-Gel and EBO-Gel.

[0016] Figure 3A-C. Oxygen supply and antioxidant properties of EBO-Gel. (A) Illustration of oxygen supply and anti-inflammation mechanisms offered by EBO-Gel. (B) Oxygen release curve monitored within 10 hours. (C) H2O2 consumption capacity of EBO-Gel (n=3). Data are shown as mean ± SD.

[0017] Figure 4A-C. Enhanced intracellular exosome delivery. (A) Colocalization efficiency of different treatments (n = 3). (B) Illustrations of the procedures for the evaluation of exosome recycling. (C) Quantification of exosome recycling in the culture medium (n = 3).

[0018] Figure 5A-C. Biocompatibility and hemostatic properties assessment. (A) Cell viability of HDF-a cells incubated with different concentrations of Exo-Gel, ONB-Gel, and EBO-Gel (n=5). (B) Blood compatibility evaluated by hemolysis assay (n=3). Positive control (Triton); Blank-Gel; ExoGel; ONB-Gel; EBO-Gel. (C) Quantitation of blood loss in (B) (n=3). Data are shown as mean ± SD.

[0019] Figure 6A-E. Evaluation of enhanced cell proliferation, migration, and angiogenesis. (A) Cell proliferation assay of HDF-a cells with different treatments (n=3). (B) In vitro wound closure rate followed by scratching assay in (D) (n=3). (C) Brightfield images and quantitative result of transwell migration of HDF-a cells (n=3). Scale bar: 200 pm. Quantitative results of (D) number of branches and (E) total branches length (n=3). Data are shown as mean ± SD.

[0020] Figure 7A-D. In vivo efficacy of EBO-Gel in a rat full-thickness wound model. (A) Illustration of wound creation and treatment timeline. (B) Representative digital photos of wounds with varied treatments at different time points. Scale bar = 7mm. (C) wound closure rate (n = 6 / group) over time. (D) Relative body weight of rats after varied treatments. Figure 8A-F. Histological analysis of wounds that underwent treatments with different hydrogels. Representative images of (A) H&E and (B) Masson’s tri chrome staining of the wound tissue on Day 14. Red arrows: new blood vessel formation; Black arrows: inflammation area; Yellow arrows: newly generated-hair follicles. Scale bar: 500 pm for the normal-sized image, and 250 pm for the magnified image. Various parameters for wound healing evaluation, including: (C) Scar index; (D) Dermis thickness; (E) Collagen volume fraction; and (F) Epidermis thickness (n = 3).

[0021] Figure 9. Particle concentrations of EBO. (n = 5, mean ± SD).

[0022] Figure 10. EBO nanoparticle release profile from EBO-Gel within 48 hours at a pH of 7.4, 37 °C. (n = 3, mean ± SD).

[0023] Figure 11. In vitro degradation rate of EBO-Gel within 3 days in PBS at 37 °C. (n = 3, mean ± SD).

[0024] Figure 12. Oxygen concentration profile of EBO-Gel and Control-Gel (Blank-Gel with oxygenated exo / BSA / Dex solution) in 48 hours.

[0025] Figure 13. Quantitation of blood loss of various treatments, (n = 3, mean ± SD).

[0026] Figure 14. Quantitative analysis of hair follicle counts per high-power field (HPF) (n = 3, mean ± SD).

[0027] Figure 15. Quantitative analysis of gland count (sebaceous glands and sweat glands) per high- power field (HPF) (n = 3, mean ± SD).

[0028] Figure 16A-C. Quantification of fluorescent intensity of (A) CD31 and (B) DHE (n = 3, mean ± SD). (C) Quantification of the percentage of Ml macrophages (n = 3, mean ± SD).

[0029] Figure 17. Quantification of the percentage of M2 macrophages (n = 3, mean ± SD).

[0030] Figure 18. Quantification of fluorescent intensity of IL-6 staining (n = 3, mean ± SD).

[0031] Figure 19A-D. H&E staining of skin and subcutis tissues of (A) and (B) normal group, (C) and (D) EBO-Gel injected group at Day 3 post-injection. Scale bar = 500 pm (A and C), 100 pm (B and D).

[0032] Figure 20. (A) Crosslinking mechanisms and structure of EBO-Gel. (B) Schematic of the preparation of ONB and EBO.

[0033] DETAILED DESCRIPTION

[0034] Wound healing is a prominent clinical concern that can be hindered by inadequate angiogenesis, inflammation, and chronic hypoxia. Hypoxia plays a pivotal role in the delayed healing process. While exosomes derived from adipose tissue-derived stem cells have shown promise in accelerating wound healing by carrying several growth factors and microRNAs to promote angiogenesis, it has been observed that intracellular cargo delivery of exosomes is compromised in hypoxic tissues due to activated hypoxia-induced endocytic recycling. To address this challenge, we have developed a novel strategy involving the coating of oxygen nanobubbles with exosomes and incorporating them into a hybrid hydrogel, such as a polyvinyl alcohol (PVA) / gelatin hybrid hydrogel. See Han et al., Nature Communications, Vol. 15, No. 1, 3435, 2024 and its Supplementary Information, which are incorporated herein by reference.

[0035] The hydrogel design not only mitigates wound hypoxia but most importantly provides for the efficient delivery of exosomal nanoparticles, which to date has been confounded by very low, to lack of, uptake by cells in hypoxia. Furthermore, the self-healing properties of the hydrogel, along with its component, gelatin, aids in hemostasis, thereby benefiting acute and surgical wound healing. Additionally, the crosslinking bonds within the hydrogel facilitate the decomposition of hydrogen peroxide (H2O2), alleviating wound inflammation. Overall, the multifunctional hydrogel demonstrates the ability to enhance wound healing through increased angiogenesis, enhanced exosome delivery, hypoxia mitigation, and inflammation inhibition.

[0036] Definitions.

[0037] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley ’s Condensed Chemical Dictionary 14thEdition, by R. J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

[0038] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0039] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0040] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted.

[0041] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

[0042] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the terms "about" and "approximately" are intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The terms "about" and "approximately" can also modify the endpoints of a recited range as discussed above in this paragraph.

[0043] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible subranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0044] This disclosure provides ranges, limits, and deviations to variables such as volume, mass, percentages, ratios, etc. It is understood by an ordinary person skilled in the art that a range, such as “number 1” to “number ”, implies a continuous range of numbers that includes the whole numbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4, 5, ... 9, 10. It also means 1.0, 1.1, 1.2. 1.3, ... , 9.8, 9.9, 10.0, and also means 1.01, 1.02, 1.03, and so on. If the variable disclosed is a number less than “numberlO”, it implies a continuous range that includes whole numbers and fractional numbers less than numberlO, as discussed above. Similarly, if the variable disclosed is a number greater than “numberlO”, it implies a continuous range that includes whole numbers and fractional numbers greater than numberlO. These ranges can be modified by the term “about”, whose meaning has been described above.

[0045] The recitation of a), b), c), ... or i), ii), iii), or the like in a list of components or steps do not confer any particular order unless explicitly stated.

[0046] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0047] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0048] An "effective amount" refers to an amount effective to treat a disease, disorder, and / or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect. Alternatively, the terms "effective amount" or "therapeutically effective amount," as used herein, refers to a sufficient amount of an agent or a composition or combination of compositions being administered that will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study. The dose could be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration of the compositions, the type or extent of supplemental therapy used, ongoing disease process and type of treatment desired (e.g., aggressive vs. conventional treatment).

[0049] The terms "treating", "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition (e.g., a scar) from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development (e.g., inhibiting the formation of a scar or reducing its severity); (iii) relieving the disease, pathologic or medical condition (e.g., a wound); and / or (iv) diminishing symptoms associated with the disease, pathologic or medical condition (e.g., the propensity of a wound to bleed). Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping, or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and / or prophylactic administration, as appropriate.

[0050] As used herein, "subject" or “patient” means an individual having symptoms of, or at risk for, a disease or other malignancy. A patient may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, the patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the methods provided herein, the mammal is a human.

[0051] As used herein, the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of a compound of the disclosure into a subject by a method or route that results in at least partial localization of the compound to a desired site. The compound can be administered by any appropriate route that results in delivery to a desired location in the subject.

[0052] The compound and compositions described herein may be administered with additional compositions to prolong stability and activity of the compositions, or in combination with other therapeutic drugs.

[0053] The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.

[0054] The term “substantially” as used herein, is a broad term and is used in its ordinary sense, including, without limitation, being largely but not necessarily wholly that which is specified. For example, the term could refer to a numerical value that may not be 100% the full numerical value. The full numerical value may be less by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

[0055] Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of’ or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of' excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of' and "consisting of' may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations, that is not specifically disclosed herein.

[0056] This disclosure provides methods of making the compounds and compositions of the invention. The compounds and compositions can be prepared by any of the applicable techniques described herein, optionally in combination with standard techniques of organic synthesis. Many techniques such as etherification and esterification are well known in the art. However, many of these techniques are elaborated in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr., 1984; and Vol. 6; as well as standard organic reference texts such as March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Ed., by M. B. Smith and J. March (John Wiley & Sons, New York, 2001); Comprehensive Organic Synthesis. Selectivity, Strategy & Efficiency in Modem Organic Chemistry. In 9 Volumes, Barry M. Trost, Editor-in-Chief (Pergamon Press, New York, 1993 printing); Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Cary and Sundberg (1983); for heterocyclic synthesis see Hermanson, Greg T., Bioconjugate Techniques, Third Edition, Academic Press, 2013.

[0057] The term “heteroatom” refers to any atom in the periodic table that is not carbon or hydrogen. Typically, a heteroatom is O, S, N, P. The heteroatom may also be a halogen, metal, or metalloid.

[0058] As used herein, the term "substituted" or “substituent” is intended to indicate that one or more (for example, in various embodiments, 1-10; in other embodiments, 1-6; in some embodiments 1, 2, 3, 4, or 5; in certain embodiments, 1, 2, or 3; and in other embodiments, 1 or 2) hydrogens on the group indicated in the expression using “substituted” (or “substituent”) is replaced with a selection from the indicated group(s), or with a suitable group known to those of skill in the art, provided that the indicated atom’s normal valency is not exceeded, and that the substitution results in a stable compound.

[0059] Substituents of the compounds and polymers described herein may be present to a recursive degree. In this context, "recursive substituent" means that a substituent may recite another instance of itself. Because of the recursive nature of such substituents, theoretically, a large number may be present in any given claim. One of ordinary skill in the art of organic chemistry understands that the total number of such substituents is reasonably limited by the desired properties of the compound intended. Such properties include, by way of example and not limitation, physical properties such as molecular weight, solubility or log P, application properties such as activity against the intended target, and practical properties such as ease of synthesis. Recursive substituents are an intended aspect of the invention. One of ordinary skill in the art of organic chemistry understands the versatility of such substituents. To the degree that recursive substituents are present in a claim of the invention, the total number in the repeating unit of a polymer example can be, for example, about 1-5,000, about 1-1,000, about 1-500, about 1-100, about 1-50, about 1-40, about 1-30, about 1-20, about 1-10, or about 1-5.

[0060] The term “molecular weight” for the copolymers disclosed herein refers to the average number molecular weight (Mn). The corresponding weight average molecular weight (Mw) can be determined from other disclosed parameters by methods (e.g., by calculation) known to the skilled artisan.

[0061] The term "negatively expressing" a cell marker refers to a cell that lacks or does not express a particular surface protein or molecule that is typically associated with a specific cell type or stage of development.

[0062] As used herein, the term "exosomes" refers to small, naturally occurring extracellular vesicles (EVs) released by cells that act as messengers, carrying proteins, DNA, and RNA to other cells. Exosomes originate from the repeated invagination of the membrane of multivesicular bodies, which then fuse with the cell's plasma membrane, releasing the exosomes. Exosomes can be artificially created using various methods, including top-down (disrupting cells) or bottom-up (assembling from components) approaches. As discussed herein, the exosomes used in embodiments of the invention can be derived from the cells extracted or derived from cells such as those discussed herein, or cells from natural products, including plant products such as, but not limited to, ginger, Pueraria lobata, lemon, broccoli, apple and curcumin.

[0063] Embodiments of the Technology.

[0064] This disclosure provides an oxygen releasing hydrogel comprising: a) exosome coated oxygen nanobubbles, wherein an exosome coated oxygen nanobubble comprises: i) an oxygen gas nanobubble encapsulated in a glycosylated protein-conjugate shell; and ii) an outer shell comprising isolated exosomes, wherein the isolated exosomes encapsulate the glycosylated protein-conjugate shell; and b) a hybrid hydrogel comprising an ionic-crosslinked polymer, a heteroatom substituted polymer, and a crosslinker that crosslinks the heteroatom substituted polymer; wherein the exosome coated oxygen nanobubbles are substantially evenly distributed in the hybrid hydrogel to form the oxygen releasing hydrogel.

[0065] In some embodiments, the glycosylated protein-conjugate shell comprises dextran conjugated to bovine serum albumin.

[0066] In some embodiments, the glycosylated protein-conjugate shell has a weight ratio of dextran to bovine serum albumin of about 1:1, about 1.5: 1, about 2:1, about 2.5: 1, about 3 : 1 , or about 3.5: 1.

[0067] In some embodiments, the isolated exosomes are human adipose stem cell-derived exosomes. In some embodiments, the isolated exosomes are membranes that are isolated human adipose stem cell- derived exosome membranes.

[0068] In some embodiments, the isolated exosomes comprise mesenchymal stem cell markers CD44, CD90, CD105, or a combination thereof. In various embodiments, the isolated exosomes negatively express (or do not comprise) mesenchymal stem cell markers CD19, CD45, CD106, or a combination thereof.

[0069] In some embodiments, the exosome coated oxygen nanobubbles comprise a bilayer of the isolated exosomes that coat and / or encapsulate the oxygen nanobubbles, or multiple layers of the isolated exosomes that coat and / or encapsulate the oxygen nanobubbles.

[0070] In some embodiments, the crosslinker comprises borax. In various embodiments, the crosslinker comprises boronic crosslinks via borate ester bonds to heteroatoms of the heteroatom substituted polymer. In some embodiments, the heteroatom substituted polymer comprises heteroatom substituents selected from the group consisting of OR, SR, and NHR, wherein R is H or a crosslinker. In some embodiments, the heteroatom substituted polymer comprises polyvinyl alcohol (PVA), dextran, chitosan, alginate, starch, poly( / V-vinylpyrrolidone) (PVP), poly(V-isopropylacrylamide) (NIPAAm), polyethylene glycol (PEG), or a combination thereof. In one specific embodiment, the heteroatom substituted polymer is PVA. In another specific embodiment, the heteroatom substituted polymer is dextran. In some embodiments, a combination of dextran and PVA can be used as the heteroatom substituted polymer.

[0071] In some embodiments, the ionic-crosslinked polymer is a gel or gelatin.

[0072] In some embodiments, the ionic crosslinked polymer is dextran or a similar polymer such as chitosan, alginate, starch, poly(A-vinylpyrrolidone) (PVP), poly(A-isopropylacrylamide) (NIPAAm), or polyethylene glycol (PEG). In various embodiments, one or more additional ionic-crosslinked polymers can be included in the hydrogel. For example, a hydrogel can include the combination of any two or more of the above-mentioned ionic-crosslinked polymers. In one specific embodiment, a hydrogel includes the combination of gelatin and dextran.

[0073] In some embodiments, a wt.% ratio of the heteroatom substituted polymer to the ionic crosslinked polymer is about 3 : 1 to about 6:1, about 4: 1 to about 5: 1, about 3:1, about 3.5:1, about 4:1, about 4.5: 1, about 5:1, about 5.5:1, about 6: 1, or about 6.5:1.

[0074] In some embodiments, a wt.% ratio of polyvinyl alcohol to gelatin is about 3:1 to about 6:1, about 4:1 to about 5: 1, about 3:1, about 3.5: 1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, about 6:1, or about 6.5:1.

[0075] In some embodiments, a wt.% ratio of the heteroatom substituted polymer to the ionic- crosslinked polymer is about 3 : 1 to about 6:1, about 4: 1 to about 5: 1, about 3:1, about 3.5:1, about 4:1, about 4.5: 1, about 5:1, about 5.5:1, about 6: 1, or about 6.5:1.

[0076] In some embodiments, a wt.% ratio of the heteroatom substituted polymer to the ionic- crosslinked polymer to the crosslinker is about 3:1: 1 to about 6:1:1, about 4:1: 1 to about 5:1:1, about 3:1: 1, about 3.5: 1:1, about 4:1:1, about 4.5: 1:1, about 5: 1:1, about 5.5: 1:1, about 6: 1:1, or about 6.5: 1:1.

[0077] In some embodiments, the heteroatom substituted polymer comprises PVA, the ionic- crosslinked polymer is gelatin, and the crosslinker comprises borax, wherein a wt.% ratio of polyvinyl alcohol to gelatin to borax is about 3:1:1 to about 6:1:1, about 4:1: 1 to about 5:1: 1, about 3:1: 1, about 3.5: 1: 1, about 4:1:1, about 4.5: 1:1, about 5: 1:1, about 5.5: 1:1, about 6: 1:1, or about 6.5: 1: 1.

[0078] In some embodiments, the exosome coated oxygen nanobubbles have an average hydrodynamic diameter of about 180 nm to about 200 nm or about 185 nm to about 195 nm.

[0079] In some embodiments, the exosome coated oxygen nanobubbles have a mean zeta potential of about -25 mV to about -30 mV or about -20 mV to about -25 mV.

[0080] This disclosure also provides a method for healing a wound and / or for enhancing the healing of a wound comprising: a) contacting a wound and an effective amount of an oxygen releasing hydrogel described herein (e.g., administering or applying the hydrogel to the wound) to treat the wound; b) covering the wound and the oxygen releasing hydrogel with a dressing or bandage for about 1 day to about 3 days; c) cleaning the treated wound; and d) optionally repeating steps a) to c); to thereby heal the wound and / or enhance the healing of the wound.

[0081] In some embodiments, the oxygen releasing hydrogel is an adhesive, non-toxic, biocompatible, exosome coated bovine serum albumin-based oxygen nanobubble (EBO) gel that comprises: a) exosome coated bovine serum albumin-based oxygen nanobubbles, wherein an exosome coated bovine serum albumin-based oxygen nanobubble comprises: i) oxygen gas nanobubbles encapsulated in a dextran conjugated bovine serum albumin shell; and ii) an outer shell comprising isolated human adipose stem cell-derived exosomes, wherein the isolated human adipose stem cell-derived exosome encapsulate the oxygen nanobubbles; and b) a hybrid hydrogel comprising gelatin, polyvinyl alcohol, and borax that crosslinks the polyvinyl alcohol; wherein the EBO is substantially evenly distributed in the hybrid hydrogel to form the EBO gel.

[0082] In some embodiments, about 75% or more of the oxygen gas is released from the EBO gel in about 48 hours to about 72 hours to supply an effective amount of oxygen to the wound.

[0083] In some embodiments, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% of the oxygen gas is released from the EBO gel in about 24 hours, about 48 hours, about 72 hours, or about 96 hours, to supply an effective amount of oxygen to the wound.

[0084] In some embodiments, the EBO gel dissolves from environmental moisture to facilitate cleaning the treated wound.

[0085] In some embodiments, the crosslinks comprise borate ester bonds that scavenge reactive oxygen species from the wound.

[0086] In some embodiments, the EBO gel enhances cell proliferation, cell migration, angiogenesis, or a combination thereof, to facilitate healing the wound.

[0087] In some embodiments, the EBO gel increases the rate of wound healing compared to a Tegaderm™ dressing.

[0088] In some embodiments, a wound treated with an EBO gel has closed and / or has less scarring compared to a wound treated with a transparent film dressing, such as aTegaderm™ dressing.

[0089] The invention can therefore be described by the following Statements of the Invention 1-20.

[0090] Statement 1. An oxygen releasing hydrogel comprising: a) exosome coated oxygen nanobubbles, wherein an exosome coated oxygen nanobubble comprises: i) an oxygen gas nanobubble encapsulated in a glycosylated protein-conjugate shell; and ii) an outer shell comprising isolated exosomes, wherein the isolated exosomes encapsulate the glycosylated protein-conjugate shell; and b) a hybrid hydrogel comprising an ionic-crosslinked polymer, a heteroatom substituted polymer, and a crosslinker that crosslinks the heteroatom substituted polymer; wherein the exosome coated oxygen nanobubbles are substantially evenly distributed in the hybrid hydrogel to form the oxygen releasing hydrogel.

[0091] 2. The oxygen releasing hydrogel of Statement 1, wherein the glycosylated protein-conjugate shell comprises dextran conjugated to bovine serum albumin.

[0092] 3. The oxygen releasing hydrogel of Statement 1 or 2, wherein the glycosylated protein-conj ugate shell has a weight ratio of dextran to bovine serum albumin of about 2:1.

[0093] 4. The oxygen releasing hydrogel of any one of Statements 1-3, wherein the isolated exosomes are human adipose stem cell-derived exosomes.

[0094] 5. The oxygen releasing hydrogel of any one of Statements 1-4, wherein the isolated exosomes comprise mesenchymal stem cell markers CD44, CD90, and CD 105.

[0095] 6. The oxygen releasing hydrogel of Statement 5, wherein the isolated exosomes negatively express mesenchymal stem cell markers CD19, CD45, and CD106.

[0096] 7. The oxygen releasing hydrogel of any one of Statements 1 -6, wherein the crosslinker comprises borax.

[0097] 8. The oxygen releasing hydrogel of any one of Statements 1 -7, wherein the crosslinker comprises boronic crosslinks via borate ester bonds to heteroatoms of the heteroatom substituted polymer.

[0098] 9. The oxygen releasing hydrogel of any one of Statements 1-8, wherein the heteroatom substituted polymer comprises heteroatom substituents selected from the group consisting of OR, SR, and NHR, wherein R is H or a crosslinker.

[0099] 10. The oxygen releasing hydrogel of any one of Statements 1-9, wherein the heteroatom substituted polymer comprises polyvinyl alcohol.

[0100] 11. The oxygen releasing hydrogel of any one of Statements 1-10, wherein the ionic-crosslinked polymer is gelatin.

[0101] 12. The oxygen releasing hydrogel of Statement 11, wherein a wt.% ratio of polyvinyl alcohol to gelatin is about 4:1 to about 5:1.

[0102] 13. The oxygen releasing hydrogel of any one of Statements 1-11, wherein the heteroatom substituted polymer comprises PVA, the ionic-crosslinked polymer is gelatin, and the crosslinker comprises borax, wherein a wt.% ratio of polyvinyl alcohol to gelatin to borax is about 4:1:1 to about 5:1: 1.

[0103] 14. The oxygen releasing hydrogel of any one of Statements 1-13, wherein the exosome coated oxygen nanobubbles have an average hydrodynamic diameter of about 185 nm to about 195 nm, a mean zeta potential of about -20 mV to about -25 mV, or both.

[0104] 15. A method for enhancing the healing of healing a wound comprising: a) contacting a wound and an oxygen releasing hydrogel according to any one of Statements 1- 14 in a sufficient amount to effectively treat the wound; b) covering the treated wound with a dressing (e.g., a sterile medical-grade gauze (e.g., semipermeable polyurethane film, alginate, or CMC hydrofiber)) for about 1 day to about 3 days; c) cleaning the treated wound; and d) optionally repeating steps a) to c) if needed until the wound has healed.

[0105] 16. The method of Statement 15, wherein the oxygen releasing hydrogel is an adhesive, non-toxic, biocompatible, exosome coated bovine serum albumin-based oxygen nanobubble (EBO) gel that comprises: a) exosome coated bovine serum albumin-based oxygen nanobubbles, wherein an exosome coated bovine serum albumin-based oxygen nanobubble comprises: i) oxygen gas nanobubbles encapsulated in a dextran conjugated bovine serum albumin shell; and ii) an outer shell comprising isolated human adipose stem cell-derived exosomes, wherein the isolated human adipose stem cell-derived exosome encapsulate the oxygen nanobubbles; and b) a hybrid hydrogel comprising gelatin, polyvinyl alcohol, and borax that crosslinks the polyvinyl alcohol; wherein the EBO is substantially evenly distributed in the hybrid hydrogel to form the EBO gel.

[0106] 17. The method of Statement 16, wherein about 75% or more of the oxygen gas is released from the EBO gel in about 48 hours to about 72 hours to supply an effective amount of oxygen to the wound, wherein the EBO gel dissolves from environmental moisture to facilitate cleaning the treated wound.

[0107] 18. The method of Statement 16 or 17, wherein the crosslinks comprise borate ester bonds that scavenge reactive oxygen species from the wound.

[0108] 19. The method of any one of Statements 16-18, wherein the EBO gel enhances cell proliferation, cell migration, and angiogenesis to facilitate healing the wound.

[0109] 20. The method of any one of Statements 16-19, wherein the EBO gel increases the rate of wound healing compared to a Tegaderm™ transparent film dressing, wherein a wound treated with an EBO gel has closed sooner and has less scarring compared to a wound treated with the Tegaderm™ transparent film dressing.

[0110] Exosome-coated Oxygen Nanobubble-laden Hydrogel Augments Intracellular Exosome Delivery to Mitigate Hypoxia for Enhanced Wound Healing.

[0111] We developed a novel strategy utilizing ADSC-derived Exosome coated BSA-based Oxygen nanobubbles (EBO) embedded within a self-healing hydrogel matrix as a multifunctional wound dressing. The oxygen supply component, oxygen nanobubbles (ONB), is formed by encapsulating nanoscale oxygen bubbles within a glycosylated protein conjugate composed of dextran-conjugated bovine serum albumin (BSA). The utilization of glycosylated proteins offers enhanced protein characteristics, including improved thermal stability and self-assembly properties. Additionally, the glycosylated protein conjugates have been established to have free radical scavenging features and the capacity to impede oxidative deterioration, rendering them extensively useful in biomedical applications such as tumor therapy and wound healing. Simultaneously, the glycosylated protein conjugate encapsulates the bulk nanoscale oxygen bubbles produced through ultrasonic cavitation, resulting in the formation of ONB. Exosomes derived from human adipose tissue stem cells are further coated onto the surface of the ONB, resulting in the final oxygen-carrying nanosystem, which exhibits oxygen release properties and facilitates the intracellular delivery of exosomes.

[0112] To further optimize the hemostatic and antioxidant properties of wound dressings, a novel hybrid hydrogel was developed by combining polyvinyl alcohol (PVA), gelatin (GA), and borax. The hydrogel possesses dynamic crosslinking through chemical borate ester bonds, imparting them with exceptional tissue adhesion, self-healing capabilities, and shape adaptability (Li et al., Advanced Healthcare Materials 11, 2101849 (2022)). Without being held to any particular theory, it is thought that the mechanism that is responsible for the self-healing ability of the PVA / GA hydrogel is the boronic crosslinks. Two routes of intraspecies (PVA-PVA) and interspecies (PVA-GA) crosslinking include: (i) boronic-esters and (ii) ionic crosslinks (Figure 20a). Consequently, the hydrogel is well- suited for wounds that require frequent stretching and for use in parts of the body that are in constant motion and can provide protection to wounds by effectively sealing the wound site, reducing the risk of further injury or contamination. Moreover, the borate bonds present in the hydrogel exhibit the unique ability to react with hydrogen peroxide, thereby mitigating excessive inflammation. Additionally, the incorporation of gelatin, known for its hemostatic properties, further enhances the dressings' ability to promote blood clotting, facilitating the accelerated healing of traumatic and surgical wounds. However, those of skill in the art will appreciate that alternative, low toxic means of crosslinking can be used as an alternative to, or in addition to, borax. For example, low toxic crosslinkers such as carbodiimide, sodium trimetaphosphate, VV-methylene bisacrylamide, and polycarboxylic acids can be used in the preparation of the hydrogels.

[0113] By incorporating an antioxidative and hemostatic self-healing PVA / GA gel as a scaffold, it becomes feasible to integrate an oxygen-carrying nanosystem, EBO, forming a novel wound dressing to promote therapeutic exosome delivery. This advanced dressing synergistically combines multiple functionalities, including hemostasis, antioxidant activity, oxygen delivery, and enhanced exosome transport.

[0114] Synthesis and characterization of EBO. Adipose tissue serves as an indispensable reservoir of mesenchymal stem cells for applications in tissue engineering and stem cell therapy due to its convenient accessibility through minimally invasive procedures in human subjects. In this study, we isolated ADSCs from human adipose tissue and proceeded to extract and characterize exosomes derived from these ADSCs. The isolated ADSCs exhibited the characteristic spindle shape and fibroblast-like growth pattern commonly associated with mesenchymal stem cells. Furthermore, these cells demonstrated positive expression of a panel of mesenchymal stem cell marker (CD90, CD105, and CD44), while displaying negative expression of CD106, CD45, and CD19. The isolated exosomes displayed a uniform distribution of particle sizes, with an average diameter of 125.2 nm and a concentration of 7.22 x 108particles / mL measured by nanoparticle tracking analysis (NT A). The protein concentration of exosome was quantified by bicinchoninic acid (BCA) protein assay. Transmission electron microscopy (TEM) imaging depicted exosomes with a typical circular or cupshaped morphology, with a diameter of approximately 100 nm (Figure 1A).

[0115] To produce EBO, the initial step involved the preparation of ONB, which was subsequently coated with the exosome membrane. The synthesis of ONB commenced with the conjugation of BSA to dextran sulfate through a mixture followed by ultrasonication. Meanwhile, oxygen was introduced into the system throughout the ultrasonication process. The synthesis of EBO involved subjecting the exosome and ONB to ultrasonication (Figure 20B). The mechanical forces induced by ultrasonication resulted in the transient disruption of the exosome structure, allowing for the reassembly of exosomes around the ONB, forming a core-shell structure. TEM (Figure 1C) and scanning electron microscopy (SEM) imaging confirmed the presence of this core-shell structure, with EBO exhibiting a bilayer coreshell morphology and possessing a diameter ranging from approximately 150 to 200 nm. The concentration of EBO was tested by NTA as shown in Figure 9. Dynamic light scattering (DLS) analysis revealed that ONB possessed an average hydrodynamic diameter of 122.50 nm with a mean zeta potential of -40.73 mV, while EBO exhibited an average hydrodynamic diameter of 192.02 nm with a mean zeta potential of -23.30 mV (Figure 1A and IB). The negative charges had been established to stabilize the nanobubbles generated via ultrasonic cavitation. Consequently, the negatively charged EBO can effectively shield the oxygen nanobubbles from Ostwald ripening and coalescence, thereby enhancing their stability during the long-term storage, particularly in clinical applications.

[0116] The internalization of Dio-labeled EBO into HDF-a (human dermal fibroblasts, adult) cells was demonstrated through co-incubation for 6 hours. The orthogonal XZ and YZ sections of a Z-stack confirm the presence of EBOs in the cytoplasm. The effectiveness of the conjugation between dextran sulfate and BSA was evaluated by measuring the UV-vis absorbance at 294 nm and 420 nm, which correspond to the intensity of browning, indicative of early and late Maillard reaction products (MRP), respectively. The results indicated a significant increase in absorbance at both 294 nm and 420 nm after overnight mixing, compared to non-ultrasonicated BSA (Figure ID). Furthermore, ultrasonication leads to a further increase in absorbance compared to the mixture alone, confirming the formation of glycosylated protein conjugates.

[0117] To further validate the conjugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted (Figure IF). The monomer of BSA exhibited a molecular weight of approximately 66 kDa. After ultrasonication, a similar protein band at approximately 66 kDa was observed in the shell, ONB, and EBO samples, indicating the presence of BSA in these formulations. However, the protein band appeared to be slightly shifted compared to native BSA. This shift can be attributed to the increased molecular mass resulting from the conjugation between protein and polysaccharides induced by ultrasonication, indicating the successful conjugation. FTIR analysis was performed to identify functional groups of ONB (Figure IE). The absorption bands at 1650 cm’1and 1542 cm’1were attributed to distinctive amide I and amide II bands from proteins. The spectral features of dextran appeared at approximately 1200-1000 cm’1region. The intensity change observed in the spectral regions around 1650 cm’1and 1542 cm’1were ascribed to the modifications of C=O and C-N stretching vibrations originating from amide I and II, respectively, due to the Maillard reaction. An increase in band intensity was observed in the region of 3700 to 3000 corresponding to the vibrations associated with N-H and O-H groups. This enhancement can be attributed to the higher absorbance of MRP that was generated.

[0118] The extended duration of oxygen nanobubble presence during storage is crucial for efficient oxygen release. To evaluate the stability, particularly the longevity of the core-shell structure of EBO, TEM images were obtained under different storage conditions (2 days at 37 °C and 1 month at 4 °C). Despite the dissociated protein observed in the background after 1 month at 4 °C (possibly exosome fragments with negative staining), EBO successfully maintains its core-shell structure in both conditions. This resilience is advantageous for ensuring sustained oxygen supply upon subsequent use.

[0119] Characteristics ofEBO-Gel. The aqueous mixture of PVA, gelatin and EBO is a low viscosity liquid, upon the addition of 2 wt% of borax, transient cross-links rapidly form boronic ester bonds, creating a viscoelastic hydrogel within seconds. Figure 2A and 2B show evidence of these key rheological features. Known as protorheology, these images can be used for both qualitative and quantitative inference (detailed in the Examples below). For example, the tilted vial of EBO-Gel in Figure 2A is evidence of a sufficiently high viscosity to inhibit flow on the observation timescale of 5 minutes, with an implied viscosity on the order of ~ 104Pa.s at this gravitational stress of -100 Pa. At higher applied stress, the EBO-Gel readily flows, as evidenced in Figure 2B with the material extruded by hand from a syringe with characteristic flow stress - 100,000 Pa. This is evidence for dramatic shear thinning, since the material readily flows at high stress, but retains its shape at lower stress. This injectable design further facilitates conformity with the shape of a defect, enabling precise and tailored application. SEM images reveal the porous structure and the presence of nanoscale particle in EBO-Gel (Figure 2B). In contrast to the smooth surface observed in Blank-Gel, EBO-Gel exhibits a rougher surface with nanoscale particles adhering to the gel skeleton, indicating the existence of EBO. The cumulative EBO release profile was shown in Figure 10, indicating that over 80% of EBO is released from EBO-Gel within 48 hours. This corresponds with the intervals for refreshing wound dressings, ensuring the efficient release of nanoparticles to the wound bed before the next dressing. To ascertain the shape adaptability, the hydrogel underwent iterative remodeling, wherein it was repeatedly reconfigured into various morphologies including star shape, crescent moon shape, round, and cross shape, which demonstrates the favorable capacity of EBO-Gel to conform effectively to irregular wound shapes, thus highlighting its adaptability in a clinical setting.

[0120] Self-healing ability is an additional crucial factor that influences the wound adaptability of hydrogel dressings. The mechanism that is responsible for the self-healing ability of our PVA-GA hydrogels is the transient boronic crosslinks. Two possible routes were presumed, intra- (PVA-PVA) and interspecies (PVA-GA) crosslinks: (i) boronic-esters; and (ii) ionic crosslinks. We examined self- healing properties at both macro and micro levels. At the macroscopic level, two EBO-Gel specimens, each stained with a distinct color (purple and yellow), were brought together. In this protorheology demonstration, the reassembled gel was subjected to stretching after a minute to evaluate its healing capacity. To explore the self-healing characteristic, an evaluation of the gel linear viscoelasticity was assessed further carefully following a cyclic process of cutting and reassembling. The findings revealed that even after undergoing three cycles of cutting and reassembling, the hydrogels could consistently maintain their linear viscoelastic properties, i.e., G and G". The results from these rheological experiments suggest that both blank- and EBO-Gels can self-heal and retain their rheological properties even after being extremely strained, which is favorable for the clinical wound care.

[0121] Adhesion properties were investigated on materials and tissues. The adhesive properties of the hydrogel on diverse substrates, including plastic, glass, rubber, and steel. We also show that the hydrogel exhibited remarkable fixation on the finger, retaining its adhesion when the fingers were flexed within a range of 0° to 90°, providing further evidence of exceptional adaptability of EBO-Gel, particularly on body parts that exhibit a wide range of motion. The adhesion can be attributed to the formation of strong hydrogen bonds between the skin tissue and EBO-Gel. This robust adhesive feature establishes the EBO-Gel as a reliable physical sealing agent for hemostasis purposes.

[0122] Effective degradation is crucial to prevent the retention of residual hydrogel in deep wounds, that could impede the healing due to its strong adhesion and tissue adaptability. In vitro degradation analysis revealed that EBO-Gel can degrade by 80% within 3 days under simulated physiological conditions (Figure 11), ensuring compatibility with the subsequent healing process and alignment with the dressing change intervals in in vivo studies. Oxygen supply and ROS elimination assessment. To verify the oxygen release properties, oxygen concentration was monitored within 10 hours. Figure 3B demonstrates the superior oxy gensupplying capabilities of both EBO-Gel and ONB-Gel in hypoxic conditions, in comparison to ExoGel and Blank-Gel. Extended oxygen release profdes were compared with a Control-Gel formed without ultrasonication. Despite a decline after 12 hours, the dissolved oxygen in the hypoxic environment exceeded that in Control-Gel by 40 hours (Figure 12). This supports our treatment of changing dressings every 2 days in subsequent in vivo experiments to sustain optimal oxygen levels in the wound bed. Subsequently, hypoxia in human fibroblast (HDF-a) cells was investigated by employing [Ru(dpp)3]C12 (RDPP) as a cellular hypoxia indicator. Remarkably, cells incubated with EBO-Gel and ONB-Gel exhibited the low RDPP fluorescence intensity, signifying effective hypoxia mitigation, which serves to validate the exceptional cellular oxygen supply capability of EBO-Gel.

[0123] ROS scavenging properties can be ascribed to two primary factors: (1) the presence of boronic ester bonds in the gel enables it to respond to hydrogen peroxide (H2O2) and facilitate its decomposition; (2) the exosomes derived from ADSCs have been demonstrated to possess antioxidant properties (Figure 20B). Figure 3C showed a rapid reduction in H2O2 concentration within the first 30 minutes of incubation with the gel, with a continued decrease observed over the course of 240 minutes, indicating the superior ability of the gel to effectively scavenge H2O2. ROS / SOD fluorescence detection on HDF-a cells further demonstrated the ROS elimination capacities of EBO-Gel.

[0124] Exosome delivery enhancement study. The oxygen sensing pathway was found to regulate endocytosis. Previous studies have also demonstrated that intracellular cargo delivery of exosomes is undermined in hypoxic tissues. Therefore, EBO is expected to restore exosome delivery efficiency in hypoxic microenvironments by supplying oxygen. To explore the exosome delivery efficiency, HDF- a cells were co-cultured with CFSE-labeled exosomes for a duration of 8 hours. Immunofluorescence was performed to assess the colocalization between exosomes and LampII, a marker of endolysosomes. Remarkably, under hypoxic conditions, a notable decrease in the colocalization of exosomes and LampII was observed, indicating a diminished transfer of exosomes to the endolysosomes, which typically serve as sites for the release of protein cargo into the cytoplasm.

[0125] Hypoxia has been known to impact endocytic recycling via the modulation of Rabl l. Consequently, the recycling of exosomes through exocytosis may hinder the efficient release of their cargo into the cellular cytoplasm, thus compromising the delivery of therapeutic cargo. To prove this hypothesis, a period of 24-hour of internalization of CFSE-labeled ADSC-derived exosome was allowed, followed by the assessment of CFSE signals that were subsequently re-secreted into the culture medium over a 12-hour interval under distinct experimental conditions, including hypoxia, normoxia, and treatment with an oxygen-enhancing agent (EBO) (Figure 4A). The obtained results revealed a heightened presence of exosome proteins recycled to the culture medium during hypoxic incubation in contrast with both EBO-treated and normoxic groups (Figure 4C). This observation signifies the potential of EBO treatment to augment the evasion of exosomes from enhanced endocytic recycling caused by hypoxia, thereby facilitating the therapeutic cargo release within the cellular cytoplasm.

[0126] Evaluation of biocompatibility and hemostatic efficacy. To examine the biocompatibility of EBO-Gel, a cytotoxicity test and a hemolysis assay were conducted. For cytotoxicity experiments, HDF-a cells were incubated with four different concentrations of Exo-Gel, ONB-Gel, and EBO-Gel for 24 hours. As shown in Figure 5A, all groups performed high cell viability, surpassing 80%, which indicates their excellent biocompatibility. For hemolysis assay, all hydrogel groups demonstrated a remarkable blood compatibility, as evidenced by a hemolysis ratio of less than 2% (Figure 5B).

[0127] Hemostasis plays a pivotal role in the initial phase of wound healing, serving as a critical step that establishes a temporary barrier to safeguard the underlying tissues against additional harm and potential infection. Given the frequent occurrence of bleeding in both surgical wounds and traumatic injuries, advanced wound dressings should possess the ability to expedite the hemostasis process. Gelatin has gained significant attention to be a promising hemostasis material due to its similarity in composition to the extracellular matrix, enabling gelatin to effectively trigger platelet aggregation and facilitate hemostasis. In addition, PVA hydrogel as a self-healing bioadhesive hydrogel offers a promising application as an embolic hemostatic agent, allowing it to seamlessly adhere to the site of bleeding, forming a physical seal that effectively controls and manages the bleeding process. Therefore, the hemostatic function of EBO-Gel was assessed both in vitro and in vivo. EBO-Gel, when mixed with rat whole blood and blood cells at a volume ratio of 2: 1 for 1 minute, showed coagulation. This underscores its hemostatic capability, similar to commercial products including CURAD® BloodStop® Hemostatic Gauze and BleedStop™, in comparison to both the control group and a nonhemostatic gel (Carbopol hydrogel group). In the rat liver hemorrhage model, the bleeding site was promptly sealed upon application of EBO-Gel, leading to a substantial reduction in blood loss, aligned with the in vitro findings (Figure 5C, and Figure 13). Our results highlight the effective hemostatic performance of EBO-Gel, in addition to wound healing characteristics.

[0128] In vitro facilitation of proliferation, migration, and angiogenesis. Cell proliferation was assessed using HDF-a cells, which were exposed to hypoxic conditions for 24 and 48 hours after treatment with hydrogels. Notably, the EBO-Gel group exhibited a higher number of proliferating cells in comparison to the other groups, as demonstrated in Figure 6A. Furthermore, real-time monitoring of cell proliferation was conducted using a BrdU incorporation assay and cell cycle analysis. The EBO- Gel group displayed a significantly higher positive signal of BrdU and more cells in S / (G2 / M) phases compared to the other groups, indicating an increase in the newly synthesized DNA, which corroborates the ability of EBO-Gel in promoting cell proliferation.

[0129] ADSC-Exo plays a crucial role in promoting dermal fibroblast migration and wound healing by releasing MALAT1, a specific long non-coding RNA. Additionally, the modulation of Apoptosis Peptidase Activating Factor 1 (APAF1) through miR-93-3p in ADSC-Exos contributes to improved cellular viability and migration, particularly in hypoxic conditions. The migration ability of HDF-a cells under hypoxic conditions was assessed using both scratching assay and transwell migration test. In the scratching assay, cell migration was visually monitored at 0, 12, and 24 hours. Remarkably, the EBO-Gel treatment group displayed a higher wound closure rate (Figure 6B) in comparison to the other groups, indicating enhanced cell migration (Figure 7D). Similarly, the EBO-Gel group exhibited a higher number of migrated cells in the transwell after 24 hours of co-incubation (Figure 6C). These results collectively demonstrate the capacity of the EBO-Gel to promote fibroblast migration.

[0130] Angiogenesis, a pivotal process in wound healing, entails the formation of tube-like structures by endothelial cells, which progressively extend, branch, and establish interconnected networks. The establishment of an extracellular matrix (ECM) is vital for neovascularization, with collagen deposition playing a fundamental role in aortic endothelial cell migration, which is an oxygen-dependent process. Hence, oxygen supply might be a potential strategy for enhancing blood vessel formation. Moreover, EBO aids angiogenesis through exosomes containing regulators such as Nrf2 and FGF2, with exosomes promoting P-catenin activation and harboring microRNAs (miR-31, miR-125a) that modulate angiogenic pathways, collectively enhancing proangiogenic effects. In vitro tube formation capacity was evaluated using HUVECs. After 6 hours of co-incubation, the EBO-Gel-treated group developed a higher number of branches (Figure 6D) and the maximum total branch length (Figure 6E), indicating the potential of EBO-Gel to facilitate angiogenesis.

[0131] In vivo wound healing analysis. EBO-Gel showed robust tissue adhesion, oxygen release, antioxidant, and enhanced exosome delivery properties, all of which are expected to be promising for noninvasive wound closure. Therefore, the wound healing performance of the EBO-Gel was assessed in a rat full-thickness defect model. Figure 7A depicts the surgical procedure, dressing application, and healing timeline. Results show that the EBO-Gel-treated group exhibited a notably accelerated healing rate compared to the other groups, particularly during the proliferation stage from day 2 to day 10 (Figure 7B and 7C). Remarkably, the Tegaderm™ transparent film group exhibited signs of infection and inflammation on days 2 and 4, while the groups treated with the hydrogel dressings described herein showed no significant infection (Figure 7B). Throughout the treatment duration, a reduction in body weight was observed during the initial two days post-surgery, which can be attributed to the recovery from anesthesia. Subsequently, the animals exhibited a gradual weight gain, with an increase in weight observed by day 14 (Figure 7D). Additionally, after 14 days of treatment, the wounds treated with the EBO-Gel displayed excellent recovery quality, characterized by the absence of hypertrophic and keloid scars.

[0132] To comprehensively assess the therapeutic effectiveness of the different treatments, tissue samples collected on Day 4 and Day 14 were subjected to histopathological evaluation using hematoxylin and eosin (H&E) staining and Masson's trichrome staining, focusing on skin repair parameters including scar index, dermis thickness, epidermis thickness, and collagen fraction. Day 4 represents a crucial time window for the inflammation and proliferation stages of the healing process, while Day 14 can reflect the regeneration stage and healing efficacy. The EBO-Gel group exhibited a consistently regenerated epidermis, increased fibroblast proliferation, enhanced angiogenesis, and collagen fiber formation. In contrast, wounds in the Tegaderm™ transparent film and Blank-Gel groups showed incomplete closure and higher areas of inflammation infiltration.

[0133] This highlights the role of EBO-Gel in influencing the inflammation, proliferation, and early regeneration stages of healing. The staining results indicated visible wound closure and the development of newly formed epidermis across all treatment groups on Day 14 (Figure 8A and 8B). However, the EBO-Gel treated group exhibited distinct characteristics with flatter wound surfaces, demonstrating a continuous and coherent epidermis, highlighting its significant potential in preventing keloid formation and achieving scarless wound healing. Furthermore, the EBO-Gel group displayed a higher density of newly formed blood vessels and reduced inflammation areas, signifying its capability to enhance angiogenesis and possess antioxidant properties. The increased count of newly generated hair follicles and glands (Figure 14 and Figure 15), and the organized formation of granulation tissue further underscored the superior quality of healing achieved through EBO-Gel treatment.

[0134] The Scar Index (SI), representing scar size, is determined by dividing the scar area (mm2) by the corresponding average dermal thickness (mm). The calculated results showed that the group treated with EBO-Gel obtained the lowest scar index, indicating a more favorable healing outcome (Figure 7C). Complete wound healing is also marked by the repair of dermis. Dermal thickness measurements revealed that the group treated with EBO-Gel exhibited a thicker dermal layer, indicating a more robust healing process and well-repaired tissue (Figure 8D). Conversely, the thickness of the newly formed epidermis was observed to be closer to that of normal skin tissue, indicating a more balanced healing response (Figure 8F).

[0135] This thinner epidermis is advantageous in preventing the formation of thick scabs or keloids, which can otherwise impair the normal functioning of the regenerated tissue. Furthermore, Masson's tri chrome staining demonstrated collagen deposition and organization in various treatments. The EBO- Gel treated group showed an abundance of densely packed collagen fibers in magnified regions. Notably, these fibers demonstrated a more mature phenotype characterized by well-organized alignment and an intricate collagen network topology, especially in comparison to the Tegaderm™ transparent film and Blank-Gel group (Figure 8E). The quantitative assessment of collagen volume fraction aligned with the observed findings, which verified the improved collagen deposition ability of EBO-Gel.

[0136] To assess the multifaceted impact of EBO-Gel on angiogenesis and anti-inflammatory responses in vivo, we performed fluorescent staining of wound tissues on Day 14 post-treatment. The immunofluorescent staining for CD31, a marker indicating angiogenesis, exhibited heightened expression and fluorescence intensity in the EBO-Gel treated group, showcasing its potential in promoting angiogenesis (Figure 16). By incorporating ADSC-exosomes, EBO-Gel has the potential to mitigate excessive inflammation, forestalling the development of dysfunctional scars throughout the healing process. Our examination encompassed ROS levels, inflammatory cytokine expression, and macrophage phenotypes. Dihydroethidium (DELE) staining unveiled significantly lower ROS levels in wounds treated with EBO-Gel in comparison to the Tegaderm™ transparent film control and Blank- Gel (Figure 16). Notably, the heightened DELE intensity in the Tegaderm™ transparent film control and Blank-Gel groups indicates prolonged elevated ROS levels in healed tissue even after 14 days of treatment, possibly contributing to hypertrophic scar formation. In the evaluation of immune cells through immunofluorescent staining for CD86 and CD206 with F4 / 80 — markers for Ml inflammatory and M2 anti-inflammatory macrophages, respectively — EBO-Gel demonstrated lower CD86 and higher CD206 expression compared to control groups (Figure 16 and Figure 17). Furthermore, the immunofluorescent staining of interleukin-6 (IL-6), an inflammatory marker, exhibited the lowest expression in the EBO-Gel group, solidifying its efficacious anti-inflammatory effects (Figure 18).

[0137] Effective wound closure could be potentially hindered if the adaptive hydrogel fails to degrade adequately in deep wounds, underscoring the crucial importance of its degradability. For in vivo degradation assessment, the visual observation of EBO-Gel reveals a progressive reduction in size and complete degradation within a 3 -day period. H&E staining of tissues surrounding the injection sites on Day 3 post-injection revealed no discernible inflammation induced by the injection compared to normal skin and subcutis tissues (Figure 19). This indicates the in vivo biocompatibility of EBO-Gel. Autolysis characterizes the degradation of EBO-Gel, where water molecules, with unshared electron pairs, initiate the hydrolysis of boron atoms. This hydrolysis causes the gradual breakdown of the borax bond crosslinked network, weakening both borax and hydrogen bond crosslinks. Consequently, the hydrogel network progressively loosens when exposed to a water environment over time, ultimately resulting in the complete dissolution of the hydrogel. To comprehensively assess the biosafety of EBO- Gel, we conducted H&E staining on visceral slices obtained from animals treated for a 14-day period. The results revealed no obvious systemic toxicity in animals treated with EBO-Gel compared to untreated animals, validating the biosafety of EBO-Gel.

[0138] Discussion.

[0139] The healing of traumatic and surgical wounds represents a significant challenge that affects millions of individuals worldwide, placing immense strain on healthcare systems and patients alike. Among the most common complications encountered in such wounds are bleeding, poor healing, and the development of hypertrophic scarring. Adipose-derived stem cell-derived exosomes have emerged as promising therapeutic agents, boasting antioxidant and pro-angiogenic properties that promote tissue regeneration. However, the hypoxic microenvironment of poorly healing wounds can compromise their delivery efficacy.

[0140] Although acute hypoxia can stimulate initial tissue regeneration by increasing vascular endothelial growth factor (VEGF), prolonged hypoxia hinders optimal recovery. While hypoxia initiates neovascularization, it cannot sustain the process, emphasizing the importance of restoring oxygen levels after the initial acute hypoxia phase in wound healing. Moreover, lower oxygen tension during hypertrophic scar progression suggests oxygen supply as a potential strategy to expedite wound healing and prevent hypertrophic scar formation. Addressing these multifaceted challenges, our study introduces a novel tissue adhesive PVA / GA hybrid hydrogel that exhibits a remarkable hemostatic synergistic effect by incorporating the therapeutic properties of gelatin, specifically targeting irregularly shaped and bleeding wounds. Moreover, we have taken a pioneering step by embedding novel exosome-coated oxygen nanobubbles within the hydrogel, thereby elevating the delivery efficiency of exosomes and addressing hypoxia. This combination not only accelerates the wound healing process but also effectively removes excess ROS from the wound tissue, alleviating hypoxia and mitigating the risk of hypertrophic scars.

[0141] The scarless healing potential of EBO-Gel is attributed to the multifaceted functions of ADSC- Exos, enriched with microRNAs such as miR-192-5p and miR-29athat regulate fibrotic responses and keloid fibroblast migration. Moreover, EBO-Gel shows promise in addressing abnormal metabolic conditions and oxygen tension in hypoxic hypertrophic scars, offering a promising avenue for further investigation in scar management.

[0142] In summary, this research has culminated in the development of a hybrid hydrogel that possesses remarkable capabilities in hemostasis, anti-inflammation, hypoxia mitigation, and enhanced exosome delivery, which, when applied to full-thickness wounds, demonstrated an accelerated healing speed and significantly improved healing quality. Notably, EBO-Gel can also be used for diabetic chronic wound treatment and chronic wound treatment, leveraging its oxygen-supplying capability, antioxidant properties, promotion of cell migration, enhancement of vascularization.

[0143] The following Example is intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Example suggests many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

[0144] EXAMPLE

[0145] Example 1. Materials and Methods.

[0146] Materials. Bovine serum albumin, dextran sulfate sodium salt (Mw 200 kDa), poly(vinyl alcohol) (Mw 89-98 kDa), sodium tetraborate decahydrate, collagenase type I, 5-Bromo-2'- deoxyuridine were purchased from Sigma-Aldrich. MTT, CFSE, Dio, LAMP2 monoclonal antibody, HiDCFDA, propidium iodide, BrDU primary antibody, Alexa Fluor™ Plus 555 Phalloidin, CD86 primary antibody, CD206 primary antibody, CD31 primary antibody, IL-6 primary antibody, DHE were obtained from Thermo Fisher Scientific. ROS / Superoxide detection assay kit, calcein AM, DAPI were purchased from Abeam. Human mesenchymal stem cell marker antibody panel was purchased from R&D systems.

[0147] ADSCs isolation, culture, and characterization. ADSCs were isolated from discarded human adipose tissue obtained from Carle Foundation Hospital. Briefly, tissue was digested in 0.075% collagenase type 1 prepared in PBS containing 2% penicillin / streptomycin for 30 mins at 37 °C, 5% CO2, and the mixture was centrifuged at 2000 rpm for 5 mins. Then, the cell pellet was resuspended in 3 mL of DMEM supplemented with 20% FBS, 1% L-glutamine, and 1% penicillin / streptomycin, and the cell suspension was then filtered through 70 pm cell strainer. The cells were plated in a lysine coated tissue culture plate and adhered for 48 h. After the confluency reached 80%, the ADSCs were subcultured.

[0148] The isolated ADSCs were characterized by flow cytometry. The cells were incubated with human mesenchymal stem cell marker antibody panel for 30 mins at room temperature, followed by centrifugation at 300 xg for 5 mins, and the sample washed twice in 2 mL of PBS with 3% BSA. Then, the cells were incubated with secondary antibody for 30 mins in the dark and were washed and resuspended in flow cytometry staining buffer for flow cytometric analysis.

[0149] Exosome isolation and characterization. At the 3rd- 8thpassage, ADSCs were cultured in DMEM with 5% exosome-depleted FBS, and the spent media was collected after 48 h and centrifuged at 2000 xg for 10 mins at 4 °C to remove cell debris. The supernatant was then ultrafdtered with a 100 kDa filter (Ami con 15), and the resulting solution was ultracentrifuged at 120000 xg for 90 mins at 4 °C to obtain exosome pellets. The exosome was resuspended in cold PBS and stored at -80 °C and used within one month.

[0150] To characterize the isolated exosome, nanoparticle tracking analysis (NTA) NTA (NanoSight NS300, Malvern Panalytical) was used to measure the concentration and size of exosome. TEM images were obtained by Scanning Transmission Electron Microscope (S)TEM (Thermo Fisher FEI, Tecnai G2 F20 S-TWIN). Samples were negatively stained with uranyl acetate (1 w / w%).

[0151] Preparation of ONB and EBO. ONB was prepared through an ultrasonication method. Briefly, 40 mg of BSA and 80 mg of dextran sulfate were dissolved in 10 mL sterile 1 x PBS buffer (pH = 7.4), and then fully mixed using a magnetic stirrer overnight. Further, ultrasonication was conducted in ice bath with an ultrasonic cell disruptor (SFX250, Branson, USA) with 3s on and 3s off cycle, at 50% of amplitude for 7 minutes while oxygen was continuously introduced in this system. The resulting solution was fdtered by a 0.22 pm cellulose acetate fdter. Finally, ONB was collected through ultrafiltration with Amicon 15 filter (100 kDa). EBO was generated by utilizing ultrasonication to coat the exosome membrane onto the ONB. Typically, exosomes and ONBs were thoroughly mixed at the ratio of 1 :2, followed by ultrasonication. The ultrasonication process was carried out in an ice bath, with a cycle of 10 seconds on and 10 seconds off, at an amplitude of 50%, for a duration of 5 minutes. This cycle was repeated three times.

[0152] Characterization of ONB and EBO. The hydrodynamic size distribution and zeta potential of ONB and EBO was obtained by the Litesizer™ 500 (Anton Paar). (S)TEM was employed to acquire TEM images, with the samples being negatively stained with 1 w / w% uranyl acetate. The NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Catalog number: ND-ONE-W, ThermoFisher Scientific) was utilized to measure the UV-vis absorbance. The infrared spectra of BSA and ONB were obtained by a FT-IR spectrometer (Thermo-Nicolet Is-50 FTIR). Oxygen release property was monitored by Orion™ Versa Star Pro™ Dissolved Oxygen Electrochemistry Benchtop Meter (ThermoFisher Scientific) every 0.5 h in the duration of 10 h.

[0153] EBO-Gel synthesis and characterization. PVA and gelatin were completely dissolved in distilled water, resulting in the final concentrations of 8 wt% and 2 wt%, respectively. Subsequently, a 2 wt% solution of borax was added to the mixture, facilitating the formation of the blank PVA / GA gel. To prepare the Exo-Gel, ONB-Gel, and EBO-Gel formulations, exosomes, ONBs, and EBO were evenly distributed within the PVA / GA mixture (10wt% PVA and 2.5wt% GA) and were further crosslinked by an equivalent volume of a 2 wt% borax solution.

[0154] The tilted vial in Figure 2A was used to make an estimate of the flow viscosity. Using gravitational flow down an incline, an upper bound viscosity is estimated to be r] = pgh2sin0 / 2vs= 1.9 x 104Pa. s based on an assumed fluid thickness h = 10mm, tilt angle 0 = 40°, and maximum velocity based on a displacement of 5 mm over the 5 mins of observation (Journal of Rheology 68, 113-144 (2024)). This viscosity is observed at the characteristic driving stress from gravity of cr = pgh = 98 Pa.

[0155] Two methods were used to demonstrate the self-healing properties. The first method was to fragmentize the PVA-GA samples and reprocess them. To reprocess the samples, they were cut up into small pieces (about 5 X 5 mm squares) before blobbing and sandwiching them between two glass plates at 1.2 mm gap. For each reprocessing, about 10wt% of fresh sample was added to compensate for small mass lost from the fragmentation. Linear viscoelasticity (vide supra) of the gels were measured as a probing surrogate metric for both virgin (a newly crosslinked gel) and reprocessed samples. Results reveal no significant difference up to the third reprocessing, with only a slight increase in LVE attributable to evaporation. The second method was to probe the rheological recovery from large deformation. For this experiment, a small-amplitude oscillatory shear (SAGS) at the strain amplitude of 5% was first applied for 60s, and then large-amplitude oscillatory shear (LAOS) at 200% for 60s, and lastly back to SAGS at 5% to probe the recovery of linear viscoelasticity. The recovery was monitored for 30 mins until the PVA-GA gels reach a final logarithmic aging regime. Collagen precursor solutions are made and kept on ice until the test time. Approximately 55 pL of the collagen precursor solutions was deposited on the bottom plate and the upper plate was then lowered into position. A thin layer of heavy mineral oil (FCC / USP, Fisher Chemical) was applied to the sample free surface to prevent evaporation. The temperature was then raised to 37 °C to initiate gelation.

[0156] ESEM (FEI Quanta FEG 450 ESEM) was employed to visualize microstructures of hydrogels. Macroscopic self-healing property was evaluated as follows. Two EBO-Gel specimens, each with a diameter of 2.2 cm, were fabricated and subsequently subjected to staining using crystal violet and red dyes, respectively. The hydrogels were then halved, and one half from each type of the hydrogel was juxtaposed. After 1 minute, the reassembled gel was subjected to stretching to assess its healing capacity. The diffusion of dye was monitored for 100 minutes. The assessment of shape adaptability involved placing the hydrogel within various silicone molds to evaluate its ability to conform to different shapes. To demonstrate the injectability of the hydrogel, the stained hydrogel was loaded into a syringe with an inner diameter of 4.7 mm and subsequently extruded to form the shape of “UIUC”. Oxygen release within 48 hours was monitored by Orion™ Versa Star Pro™ Dissolved Oxygen Electrochemistry Benchtop Meter (ThermoFisher Scientific) using a previously developed methodology (ACS Applied Nano Materials 6, 13116-13126 (2023)).

[0157] In vitro degradation. For in vitro degradation analysis, EBO-Gels were submerged in a 5 mL PBS solution (pH = 7.4) with lysozyme (1000 U / mL). The incubation was in a constant-temperature incubator set at 37 °C. The hydrogels were taken out for lyophilization and weighed at intervals of 0, 1, 2, and 3 days. To maintain enzyme functionality, the lysozyme solution was refreshed daily. The in vitro degradation rate was determined using the formula:

[0158] Degradation rate = [(Wo-Wt)AVo] * 100% where Wo and Wt represented the weights of the original and remaining hydrogels, respectively.

[0159] Hemolysis assay. Fresh rat blood was obtained via cardiac puncture following euthanasia and anticoagulated using Heparin. Subsequently, erythrocytes were isolated through centrifugation at 10,000 xg for 5 minutes and washed using D-PBS. Next, 0.8 mL of diluted erythrocytes were exposed to 200 pL of Triton X-100, PBS, and different hydrogels. Following a 4-hour incubation at room temperature, the mixtures were centrifuged at 10,016g for 5 minutes, and the absorbance of the supernatant was measured at 540 nm using a microplate reader. The hemolytic ratio was calculated using the following formula: Hemolysis ratio = [OD(test)-OD(negative control)) / [OD(positive control)-OD(negative control)] and expressed as a percent.

[0160] Cell culture. HDF-a cells were cultured in fibroblast basal medium supplemented with fibroblast growth kit (ATCC). HUVECs were cultured in Endothelial Growth Medium 2 (EGM2, Lonza). All cells were cultivated at 37 °C and 5% CO2. Cell viability. 5 * 103HDF-a cells were pre-seeded into 96-well plate. After 24 h of culture, the medium was refreshed, and the HDF-a cells were co-incubated with hydrogels. MTT assay was performed to evaluate cell viability. In brief, 20 pL of the prepared 5 mg / mL MTT solution was added to each well of the plate and incubated at 37°C for 4 hours. Following the addition of 100 pL of DMSO, the plate was incubated for an additional 4 hours at 37 °C in the dark. Ultimately, the absorbance at 490 nm was measured using a microplate reader.

[0161] Cell proliferation. 2 * 103HDF-a cells were pre-seeded into 96-well plate. After 24 h of normoxia incubation, the medium was refreshed, and the hydrogels were added. Then, the well plates were kept in hypoxia chamber with a mixture of 3% O2, 5% CO2, 92% N2 at 37°C for Oh, 24 h and 48 h, respectively. At different time point, WST-1 reagent was added and the optical density at 440 nm was then measured after 4 h of incubation according to manufacturer description.

[0162] Cell migration. For scratching assay, HDF-a cells were pre-seeded in a 24-well plate. After the confluency reached 80%-90%, scratching wounds were created by 200 pL sterile pipette tips, and the culture medium was replaced with DMEM containing 1% FBS. Then, hydrogels were treated in the upper chamber of cell culture inserts (Millicell®, Sigma-Aldrich), and the plates were kept in hypoxia chamber with the hypoxic mixture as described. Cell migration was observed by microscope (ZEISS) at different timepoints and was quantified by ImageJ software.

[0163] For transwell migration assay, HDF-a cells were seeded in the upper chamber of cell culture inserts with a density of 1 x 104cells / mL, while the hydrogels were treated into the lower chamber with culture medium. After 24 hours, the cells on the upper side of film were gently removed by a cotton swab, and the cells that migrated to the lower side were stained with 0.5% crystal violet for 1 hour. The number of migrated cells were quantified by ImageJ software.

[0164] Exosome uptake. To prepare Dio-labeled exosome, EBO was incubated with Dio (10 pM) for 20 mins, followed by ultrafiltration using Ami con 15 at 1000 xg for 10 mins. HDF-a cells were preseeded in 35 mm coverslip dishes and cultured with exosome-depleted medium. After attachment, Dio-labeled EBO was added and incubated for 6 hours. The cells were fixed by 4% PFA after coincubation, and the nucleus was stained with 4',6-diamidino-2-phenylindole. Intracellular uptake was observed by Z-stack 3D confocal imaging with orthogonal views at the step size of 0.3 pm (Leica SP8).

[0165] Immunofluorescence analysis. HDF-a cells were pre-seeded in 35 mm coverslip dishes. After attachment, hydrogels were added, and the imaging dishes were transferred to hypoxia chamber under the same hypoxic conditions as described. Following the completion of hypoxia incubation, the cells were washed with PBS and subsequently fixed with 4% paraformaldehyde. This was followed by permeabilization using 0.3% Triton X-100 for an additional 10 minutes. Then, the cells were incubated with a solution of 3% BSA in PBS for 1 h at 37 °C to prevent nonspecific binding. Next, various primary antibodies (anti-BrdU, anti-Lamp2) were applied to the cells and allowed to incubate overnight at 4 °C, and the cells were then incubated with secondary antibody for 1 h at 37°C. For BrdU staining, the medium was replaced with 10 pM BrdU in culture medium before hydrogels were added. After fixation, the cells were incubated with 2M HC1 for 0.5 h for acid hydrolysis. Cytoskeleton was stained by Alexa Fluor™ Plus 555 Phalloidin for 20 mins. Images were captured by a confocal laser scanning microscope (Leica SP8) and analyzed by ImageJ software.

[0166] Tube formation assay. HUVECs for tube formation assay were used before passage 6. Typically, 100 pL of Matrigel (Coming) was added in a 96-well plate and incubate to gel at 37 °C for Ih. 10000 HUVECs were seeded into each well and hydrogels were then added. Plates were further transferred into the hypoxia chamber, and supplied with a mixture of 3% O2, 5% CO2, 92% N2 at 37 °C. After 6 h, brightfield images of tube formation were captured by Leica microscope. The number of branches and total length of branches was quantified by ImageJ software.

[0167] In vitro hemostasis assay. Heparinized rat blood and hemocytes were mixed with Control, Carbopol gel, and EBO-Gel, respectively, at the volume ratio of 1:2. After 1 minute, the vial was inversed, and the hemostasis property was observed.

[0168] In vivo wound healing study. Male Sprague-Dawley rats of 250-300 g weight (8 weeks old) were obtained from Envigo Laboratory (Indianapolis, IN). All animal studies were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee and the Division of Animal Resources at the University of Illinois. The rats were randomly sorted into five groups: NT, Blank-Gel, Exo-Gel, ONB-Gel, and EBO-Gel. The precursor solutions were prepared by thoroughly mixing 0.4 ml of PVA / GA (10 wt% PVA and 2.5 wt% GA) and 0.1 ml of nanoparticle solutions (Exo, ONB, and EBO) to achieve final concentrations of 8 wt% PVA and 2 wt% GA respectively. Hydrogels were created by combining the precursor solution with an equal volume of 2 wt% borax solution before application. Full-thickness wounds were then made on the back using a sterile disposable 8 mm diameter dermal biopsy punch (MEDLINE) to a depth of 2 mm.

[0169] The prepared hydrogels were applied to the wounds post-surgery. The wound surface was ultimately covered with a Tegaderm™ transparent film dressing (4.4 x 4.4 cm2, 3M™ Tegaderm™ 1622W) and a self-adhering elastic bandage (Equate™). In the Tegaderm™ dressing group, no hydrogels were applied. Dressings were refreshed every two days following the standard of care. The old residue hydrogels were entirely removed before applying freshly prepared hydrogels, followed by covering with new Tegaderm™ dressing and bandage, as described before. The healing process was monitored by a digital camera, and the wound area was quantified by ImageJ software.

[0170] On day 14, animals were euthanized and the wound tissues were collected. Then, the tissues were fixed in 10% formalin neutral buffered solution (Sigma- Aldrich) and further embedded in paraffin and sectioned for H&E / Masson Trichrome staining according to the manufacturer manual. The histology images were acquired by microscope. Scar Index (mm) = Scar Area (mm2) / Average Dermal Thickness (mm). Dermal thickness, epidermal thickness and scar area were quantified by NDP View 2 software. The collagen volume fraction was quantified by ImageJ software. All publications, patents, and patent documents cited herein are incorporated by reference as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, many variations and modifications may be made while remaining within the spirit and scope of the invention.

[0171] While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

Claims

What is claimed is:

1. An oxygen releasing hydrogel comprising: a) exosome coated oxygen nanobubbles, wherein an exosome coated oxygen nanobubble comprises: i) an oxygen gas nanobubble encapsulated in a glycosylated protein-conjugate shell; and ii) an outer shell comprising isolated exosomes, wherein the isolated exosomes encapsulate the glycosylated protein-conjugate shell; and b) a hybrid hydrogel comprising an ionic-crosslinked polymer, a heteroatom substituted polymer, and a crosslinker that crosslinks the heteroatom substituted polymer; wherein the exosome coated oxygen nanobubbles are substantially evenly distributed in the hybrid hydrogel to form the oxygen releasing hydrogel.

2. The oxygen releasing hydrogel of claim 1, wherein the glycosylated protein-conjugate shell comprises dextran conjugated to bovine serum albumin.

3. The oxygen releasing hydrogel of claim 2, wherein the glycosylated protein-conjugate shell has a weight ratio of dextran to bovine serum albumin of about 2:1.

4. The oxygen releasing hydrogel of claim 1, wherein the isolated exosomes are human adipose stem cell-derived exosomes.

5. The oxygen releasing hydrogel of claim 4, wherein the isolated exosomes comprise mesenchymal stem cell markers CD44, CD90, and CD105.

6. The oxygen releasing hydrogel of claim 5, wherein the isolated exosomes negatively express mesenchymal stem cell markers CD19, CD45, and CD106.

7. The oxygen releasing hydrogel of claim 1, wherein the crosslinker comprises borax.

8. The oxygen releasing hydrogel of claim 1, wherein the crosslinker comprises boronic crosslinks via borate ester bonds to heteroatoms of the heteroatom substituted polymer.

9. The oxygen releasing hydrogel of claim 1, wherein the heteroatom substituted polymer comprises heteroatom substituents selected from the group consisting of OR, SR, and NHR, wherein R is H or a crosslinker.

10. The oxygen releasing hydrogel of claim 1, wherein the heteroatom substituted polymer comprises polyvinyl alcohol.

11. The oxygen releasing hydrogel of claim 1, wherein the ionic-crosslinked polymer is gelatin.

12. The oxygen releasing hydrogel of claim 11, wherein polyvinyl alcohol and gelatin are present in a wt.% ratio of about 4: 1 to about 5: 1.

13. The oxygen releasing hydrogel of claim 1, wherein the heteroatom substituted polymer comprises PVA, the ionic-crosslinked polymer is gelatin, and the crosslinker comprises borax, wherein polyvinyl alcohol, gelatin, and borax are present in a wt.% ratio of about 4:1:1 to about 5:1: 1.

14. The oxygen releasing hydrogel of claim 1, wherein the exosome coated oxygen nanobubbles have an average hydrodynamic diameter of about 185 nm to about 195 nm or a mean zeta potential of about -20 mV to about -25 mV.

15. A method for enhancing the healing of a wound comprising: a) contacting a wound and an effective amount of an oxygen releasing hydrogel according to any one of claims 1-14 to treat the wound; b) covering the wound and the oxygen releasing hydrogel with a dressing for about 1 to 3 days; c) cleaning the wound; and d) optionally repeating steps a) to c); to thereby enhance the healing of the wound.

16. The method of claim 15, wherein the oxygen releasing hydrogel is an adhesive, non-toxic, biocompatible, exosome coated bovine serum albumin-based oxygen nanobubble (EBO) gel that comprises: a) exosome coated bovine serum albumin-based oxygen nanobubbles, wherein an exosome coated bovine serum albumin-based oxygen nanobubble comprises: i) oxygen gas nanobubbles encapsulated in a dextran conjugated bovine serum albumin shell; and ii) an outer shell comprising isolated human adipose stem cell-derived exosomes, wherein the isolated human adipose stem cell-derived exosome encapsulate the oxygen nanobubbles; andb) a hybrid hydrogel comprising gelatin, polyvinyl alcohol, and borax that crosslinks the polyvinyl alcohol; wherein the EBO is substantially evenly distributed in the hybrid hydrogel to form the EBO gel.

17. The method of claim 16, wherein about 75% or more of the oxygen gas of the oxygen gas nanobubbles is released from the EBO gel in about 48 hours to about 72 hours to supply an effective amount of oxygen to the wound, and wherein moisture from the local environment dissolves the EBO gel to facilitate healing and / or cleaning the treated wound.

18. The method of claim 17, wherein the crosslinks comprise borate ester bonds that scavenge reactive oxygen species from the wound.

19. The method of claim 17, wherein the EBO gel enhances cell proliferation, cell migration, and angiogenesis to facilitate healing the wound.

20. The method of claim 17, wherein the EBO gel increases the rate of wound healing compared to a transparent fdm dressing, wherein a wound treated with an EBO gel has closed and has less scarring compared to a wound treated for the same amount of time with the transparent film dressing.

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