Systems and methods for soluble particles

Abstract

The present disclosure generally relates to systems and methods for soluble particle, e.g., micrometer or sub-micrometer- sized particles s. Certain embodiments are generally directed to relatively small particles or microbubbles that can be administered to a subject, e.g., to treat or prevent hypoxia. The particles may include a shell surrounding a gas core, e.g., containing air or oxygen. In some cases, the particles may be highly soluble, for example, such that they can dissolve following administration to a subject. In some embodiments, the particles may comprise polymers such as dextran, starch, and / or other materials. In some cases, the polymer may have a molecular weight of less than 12 kDa, and / or the polymer may be succinylated and / or acetylated. Other embodiments are generally directed to methods of making or using such particles, methods of administrating such particles to a subject, pharmaceutical compositions or kits comprising such particles, or the like.

Description

[0001] SYSTEMS AND METHODS FOR SOLUBLE PARTICLES

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 706,473, filed October 11, 2024, entitled “Systems and Methods for Soluble Particles,” by Kheir, el al., incorporated herein by reference in its entirety.

[0004] GOVERNMENT FUNDING

[0005] This invention was made with government support under HL141818 awarded by National Institutes of Health, and under W81XWH- 19- 1-0237 awarded by the Department of Defense. The government has certain rights in the invention.

[0006] FIELD

[0007] The present disclosure generally relates to systems and methods for soluble particles.

[0008] BACKGROUND

[0009] Hypoxemia, or low blood oxygen content, can occur in the setting of lung disease or airway obstruction, which causes blood to circulate through the lungs and then to the body without being fully reoxygenated. In hospitalized patients, severe, episodic hypoxemia can result from endotracheal tube occlusion (e.g. secretions), progressive lung injury, and a number of other causes. Such episodes may be addressed with airway clearance, lung recruitment, increased ventilatory support, and when needed inhaled nitric oxide or extracorporeal membrane oxygenation. However, sometimes hypoxemia is temporarily so severe and refractory to these maneuvers that myocardial contractility fails, resulting in pulseless electrical activity or even asystole; this clinically manifests as cardiac arrest.

[0010] Of note, 15-40% of in-hospital cardiac arrests (IHCA) are caused by respiratory insufficiency. Such patients exhibit a hypoxic insult compounded by an ischemic insult (i.e., cessation of cardiac activity and blood flow), resulting in severe ischemic injury to the brain, kidneys, and other organs. Survival to hospital discharge following IHCA approximates 20%, with ~1 in 3 patients suffering from substantial neurologic impairment. It is well recognized that survival following IHCA is enhanced by maneuvers that optimize oxygen delivery during IHCA (such as high-quality CPR), achieving early return of spontaneous circulation, and rapid cannulation to ECMO during CPR. Successful resuscitation from IHCA requires the rapid identification and reversal of the underlying cause, however, reversing refractory hypoxemia remains an unmet challenge.

[0011] The intravenous administration of oxygen (IVO2) via an injectable gas carrier offers a mechanism to rapidly increase blood oxygen saturation. IVCh.is distinct from blood

[0012] #14356316vl substitutes made of perfluorocarbon or hemoglobin analogs, which are optimized as circulating gas carriers in the setting of a functional lung unit rather than administering oxygen to the bloodstream. Similarly, the transfusion of oxygenated blood itself is an impractical treatment for hypoxemia because the relatively low gas fraction of blood would require administration of a volume that would quickly overwhelm the circulatory system and cause lung injury and heart failure. Thus, alternative materials have been sought to develop gas carriers that improve oxygen carrying capacity in fluids.

[0013] Several groups have described microbubbles coated by phospholipids or polymer shells to deliver oxygen to acutely reverse hypoxemia in animal models. However, translating IVO2 from biomaterials research to a clinically viable therapeutic faces many hurdles. This is primarily because the intravenous administration of even small volumes of gas in an emergency setting requires that concerns of microvascular obstruction, material-related toxicities, and product viability be addressed. It is also unknown whether the administration of IVO2 in severe hypoxemia would be clinically beneficial. Accordingly, improvements in such technologies are still needed.

[0014] SUMMARY

[0015] The present disclosure generally relates to systems and methods for soluble particles. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0016] One aspect is generally drawn to a composition. In one set of embodiments, the composition comprises particles having a shell surrounding a gas core. The particles may have an average size of less than 10 micrometers. The shell may comprise cross-linked dextran having a molecular weight of less than 60 kDa, less than 40 kDa, or less than 12 kDa. In some cases, the dextran is succinylated and / or acetylated.

[0017] The composition, in another set of embodiments, is generally directed to particles having a shell surrounding a gas core, where the particles have an average size of less than 10 micrometers. In some embodiments, the shell comprises a water-soluble polymer that exhibits cross-linking at a pH of less than 6.

[0018] Another aspect is generally drawn to a method. In one set of embodiments, the method comprises creating bubbles of gas in a solution comprising a succinylated and / or acetylated dextran having a molecular weight of less than 60 kDa, less than 40 kDa, or less than 12 kDa, and acidifying the solution to a pH of less than 7 to absorb the dextran onto the bubbles of gas and cause cross-linking of the dextran.

[0019] #14356316vl In another set of embodiments, the method comprises creating bubbles of gas in a solution comprising a water-soluble polymer having a molecular weight of less than 12 kDa, and acidifying the solution to a pH of less than 7 to absorb the water-soluble polymer onto the bubbles of gas.

[0020] Several methods are disclosed herein of administering a subject with a composition for prevention or treatment of a particular condition. It is to be understood that in each such aspect of the disclosure, the disclosure specifically includes, also, the compound for use in the treatment or prevention of that particular condition, as well as use of the compound for the manufacture of a medicament for the treatment or prevention of that particular condition.

[0021] In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, microbubbles such as those described herein. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, microbubbles such as those described herein.

[0022] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0025] Figs. 1A-1H illustrate certain microbubbles for carrying gas, in accordance with one embodiment;

[0026] Figs. 2A-2G illustrate the dissolution of certain microbubbles, in another embodiment;

[0027] Figs. 3A-3L illustrate the dissolution of certain microbubbles following administration, in still another embodiment;

[0028] Figs. 4A-4S illustrate swine experiments using certain microbubbles in yet another embodiment; and

[0029] #14356316vl Figs. 5A-5W illustrate rodent experiments using certain microbubbles in still another embodiment.

[0030] DETAILED DESCRIPTION

[0031] The present disclosure generally relates to systems and methods for soluble particles, e.g., micrometer or sub-micrometer- sized particles. Certain embodiments are generally directed to relatively small particles or microbubbles that can be administered to a subject, e.g., to treat or prevent hypoxia. The particles may include a shell surrounding a gas core, e.g., containing air or oxygen. In some cases, the particles may be highly soluble, for example, such that they can dissolve following administration to a subject. In some embodiments, the particles may comprise polymers such as dextran, starch, and / or other materials. In some cases, the polymer may have a molecular weight of less than 12 kDa, and / or the polymer may be succinylated and / or acetylated. Other embodiments are generally directed to methods of making or using such particles, methods of administrating such particles to a subject, pharmaceutical compositions or kits comprising such particles, or the like.

[0032] One aspect is generally drawn to particles having a shell surrounding a gas core. The gas may be, for example, air or oxygen. The shell may include a polymer that, when administered to a subject, can readily solubilize into components that can be excreted or removed from the subject’s body (e.g., via the kidneys, hepatic clearance, etc.). Such polymers may represent a lower safety risk, and may be desirable for some applications.

[0033] While other work has used polymers such as dextran or starch to encapsulate a gas, such work has typically used relatively high molecular weight or hydrophobic (i.e., poor water solubility) polymers, e.g., polymers having molecular weights of 70 kDa, or more, or polymers that form relatively large nanoparticle aggregates in solution that cannot be excreted effectively in vivo. Such polymers were generally believed to be more stable, e.g., when administered to a subject. In contrast, as discussed herein, certain aspects of the present disclosure are generally directed to low molecular weight polymers that have been modified or derivatized in certain ways to improve their water solubility and / or their ability to be excreted or removed from the body. For instance, in some cases, the polymers may have molecular weights of less than 12 kDa, or other values such as any of those described herein. Thus, such particles are generally highly water-soluble, and are not particularly stable after administration to a subject.

[0034] For example, in some embodiments, dextran may be modified by adding charged groups, such as acetyl groups and / or succinyl groups, and / or zwitterionic groups. These

[0035] #14356316vl groups may improve the solubility of dextran in water, e.g., due to the presence of the additional charges on the molecule, and / or diminish undesirable biologic interactions. In some cases, a relatively high degree of acetylation and / or succinylation may be present. For instance, the dextran may exhibit a substitution of between 0 and 2, or between 1 and 3, etc., of acetyl groups and / or succinyl groups. Dextran has a maximum substitution value of 3, i.e., unmodified dextran has 3 available OH groups that can be modified, e.g., acetylated and / or succinylated. Dextrans having relatively high substitutions have not previously been studied for use in such applications, as it was believed that such high charge states could destabilize the particle. In addition, functional groups such as acetyl groups and / or succinyl groups, carboxylic acid group, zwitterionic groups, etc. may readily allow the dextran to become cross-linked in certain embodiments, e.g., upon exposure to acidic conditions (for example, a pH of 6 or less), thereby promoting stability of particles comprising such dextrans (e.g., prior to administration to a subject), without using relatively high molecular weight dextrans to promote stability of the particles.

[0036] In some embodiments, particles having a shell surrounding a gas core may be administered to a subject, such as a human subject. For example, the subject may be one who has or is at risk of hypoxia, e.g., a condition in which the subject experiences inadequate supply of oxygen to the tissues. Hypoxia may be caused by a range of conditions, such as stroke, acute trauma, cardiac arrest, exposure to carbon monoxide, or other conditions such as those described herein. It is believed that by administering particles having a shell surrounding a gas core, e.g., as described herein, hypoxia may be prevented or treated.

[0037] The above discussion is a non-limiting example of one embodiment of the present disclosure that is generally directed to particles having a shell surrounding a gas core. However, other embodiments are also possible. Accordingly, more generally, various aspects are directed to various systems and methods for soluble microbubbles.

[0038] Certain aspects are generally directed to particles, for example, microparticles or nanoparticles. In some cases, the particles may be formed from a shell surrounding a gas. The gas may thus be present as a core within the particle. As discussed herein, in some embodiments, the particles may be present within a composition that is administrable to a subject, although in other embodiments, the particles may be used for applications that do not involve the treatment of a subject.

[0039] The gas contained within a particle may, in some embodiments, comprise air and / or oxygen. Different particles may have the same or different gases contained therein. In some cases, the gas is air, or another gas, enriched with oxygen. For example, a gas within a

[0040] #14356316vl particle may contain at least at least 25 vol%, at least 30 vol%, at least 35 vol%, at least 40 vol%, at least 45 vol%, at least 50 vol%, at least 55 vol%, at least 60 vol%, at least 65 vol%, at least 70 vol%, at least 75 vol%, at least 80 vol%, at least 85 vol%, at least 90 vol%, or at least 95 vol% 02. In some cases, the gas contained within a particle is substantially pure 02. In some embodiments, the gas core comprises, essentially consists of, or consists of oxygen.

[0041] In addition, it should be understood that in other embodiments, other gases may be used, e.g., in addition and / or instead of air and / or O2. For example, a particle may contain gases such as carbon dioxide, carbon monoxide, nitrogen, nitric oxide, nitrous oxide, an inhalational anesthetic, hydrogen sulfide, argon, helium, xenon, etc. In some embodiments, the gas core can comprise hydrogen gas, ozone, and / or other gases that are appropriate for other applications such as gas-based antimicrobial agents. One or more of these and / or other gases may be present in various embodiments. In some embodiments, the gas is not a perfluorocarbon. The gas may be present at ambient pressures (e.g., about 1 atm) in some cases. However, in some embodiments, a gas may be present at higher pressures, e.g., at least 1.1 atm, at least 1.2 atm, at least 1.5 atm, at least 2 atm, etc.

[0042] In addition, in some embodiments, the particles may contain relatively high amounts or concentrations of gas. For example, in some cases, a particle may have at least 20 vol%, at least 30 vol%, at least 40 vol%, at least 50 vol%, at least 60 vol%, at least 70 vol%, at least 80 vol%, at least 90 vol%, or at least 95 vol% of a gas. In some embodiments, a particle may have no more than 95 vol%, no more than 90 vol%, no more than 80 vol%, no more than 70 vol%, no more than 60 vol%, no more than 50 vol%, no more than 40 vol%, no more than 30 vol%, or no more than 20 vol% of a gas. Combinations of any of these are also possible in certain cases; for example, a particle may contain between 20 vol% and 30 vol% gas, between 50 vol% and 70 vol% gas, between 40 vol% and 60 vol% gas, etc.

[0043] In some cases, a particle may contain at least 20 ml of a gas, per gram of polymer. In certain cases, a particle may have at least 21 ml / g, at least 22 ml / g, at least 23 ml / g, at least 24 ml / g, at least 25 ml / g, at least 26 ml / g, at least 27 ml / g, at least 28 ml / g, at least 29 ml / g, at least 30 ml / g, at least 31 ml / g, at least 32 ml / g, at least 33 ml / g, at least 34 ml / g, at least 35 ml / g, at least 36 ml / g, at least 37 ml / g, at least 38 ml / g, at least 39 ml / g, at least 40 ml / g, etc. of gas per particle. In some cases, the particle may have at least 45 ml / g, at least 50 ml / g, at least 55 ml / g, at least 60 ml / g, at least 65 ml / g, at least 70 ml / g, at least 75 ml / g, at least 80 ml / g, at least 85 ml / g, at least 90 ml / g, at least 95 ml / g, at least 100 ml / g, etc. of gas per particle. In some embodiments, the particle may have no more than 100 ml / g, no more than 95 ml / g, no more than 90 ml / g, no more than 85 ml / g, no more than 80 ml / g, no more than 75

[0044] #14356316vl ml / g, no more than 70 ml / g, no more than 65 ml / g, no more than 60 ml / g, no more than 55 ml / g, no more than 50 ml / g, no more than 45 ml / g, etc. of gas per particle. In some cases, the particle may have no more than 40 ml / g, no more than 39 ml / g, no more than 38 ml / g, no more than 37 ml / g, no more than 36 ml / g, no more than 35 ml / g, no more than 34 ml / g, no more than 33 ml / g, no more than 32 ml / g, no more than 31 ml / g, no more than 30 ml / g, no more than 29 ml / g, no more than 28 ml / g, no more than 27 ml / g, no more than 26 ml / g, no more than 25 ml / g, no more than 24 ml / g, no more than 23 ml / g, no more than 22 ml / g, no more than 21 ml / g, or no more than 20 ml / g, etc. of gas per particle. Combinations of any of these ranges are also possible in certain cases.

[0045] In various aspects, the particles may have an average diameter of, for example, less than 20 micrometers, less than 15 micrometers, less than 10 micrometers, less than 5 micrometers, less than 3 micrometers, less than 2 micrometers, less than 1 micrometer, less than 0.5 micrometers, less than 0.3 micrometers, less than 0.2 micrometers, less than 0.1 micrometers, etc. In some cases, the particle may have an average diameter of at least 0.1 micrometers, at least 0.2 micrometers, at least 0.3 micrometers, at least 0.5 micrometers, at least 1 micrometer, at least 2 micrometers, at least 3 micrometers, at least 5 micrometers, at least 10 micrometers, at least 15 micrometers, at least 20 micrometers, etc. Combinations of any of these are also possible. For example, the particles may have an average diameter of between 5 micrometers and 10 micrometers, between 1 micrometer and 5 micrometers, between 0.5 micrometers and 2 micrometers, between 0.5 micrometers and 3 micrometers, between 0.2 micrometers and 2 micrometers, between 3 micrometers and 15 micrometers, etc. In addition, the particles may be spherical and / or non-spherical.

[0046] In some embodiments, the particles may have an average shell thickness of less than 500 nm, less than 300 nm, less than 200 nm, less than 100 nm, less than 50 nm, less than 30 nm, less than 20 nm, less than 10 nm, less than 5 nm, less than 3 nm, less than 2 nm, etc. In some embodiments, the particles may have an average shell thickness of at least 1 nm, at least 2 nm, at least 3 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 500 nm, etc. Combinations of these ranges are also possible. For example, the particles may have an average shell thickness of between 10 nm and 200 nm, between 10 nm and 100 nm, between 10 nm and 50 nm, between 15 nm and 200 nm, between 15 nm and 100 nm, between 15 nm and 50 nm, between 20 nm and 200 nm, between 20 nm and 100 nm, between 20 nm and 50 nm, between 20 nm and 40 nm, between 30 nm and 40 nm, between or 20 nm and 30 nm, etc. In addition,

[0047] #14356316vl the shell thickness of a particle may be uniform, or non-uniform in some cases (for example, if the gas core is not concentrically located within the particle).

[0048] In one set of embodiments, the particle may comprise a shell comprising a water- soluble polymer, such as dextran, starch, or other polymers such as any of those described herein. Without wishing to be bound by any theory, it is believed that such polymers may be readily solubilized once administered to a subject, releasing the gas trapped inside the particle, and / or such polymers may allow for the supersaturation of gas within the environment in which the particles are administered. In addition, the resulting products from the particle (e.g., the polymers forming the shell) may be more easily excreted or removed from the subject’s body (e.g., via the kidneys, hepatic clearance, etc.), due to their water solubility.

[0049] In some cases, the water solubility of a polymer present with an particle shell may be determined by determining its average size or hydrodynamic radius when the polymer is placed in water (e.g., pure water at a pH of 7, or in some cases, in saline or a physiological fluid, such as blood). In some cases, the polymer may be one that exhibits a mean or average hydrodynamic radius of less than 50 nm, less than 30 nm, less than 20 nm, less than 15 nm, or less than 10 nm, e.g., as determined by techniques such as laser light scattering. In some cases, the polymer may be sufficiently soluble in water that its hydrodynamic radius in solution cannot be accurately detected or determined. In addition, in certain cases, the polymer may have relatively high water solubility, for example, a solubility of at least 5 mg / ml, at least 10 mg / ml, or at least 15 mg / ml, etc.

[0050] In some embodiments, the polymer may be one that is amphiphilic. Polymers such as dextran or starch may be rendered amphiphilic by modifying some or all of the hydroxyl groups on the dextran or starch with other chemical functional groups, such as succinyl groups, acetyl groups, alkyl groups (such as carboxylic groups), cationic or anionic groups, zwitterionic groups (e.g., n-oxide groups), phosphorylcholine, sulfobetaine, carboxybetaine, or the like. For example, dextran has 3 hydroxyl groups per polymer repeat. In some embodiments, the degree of substitution of the hydroxyl groups of dextran with another chemical functional group may be at least, at least 1, at least 1.5, at least 2, or at least 2.5. In some cases, the degree of substitution may be at most 3, at most 2.5, at most 2, at most 1.5, at most 1, or at most 0.5. In certain cases, combinations of any of these are possible, e.g., the substitution of dextran may be between 1 and 3, between 0 and 2, between 1.5 and 2.5, etc. If more than one chemical functional group is present, the degree of substitution of each may each be independent, and may be between any of the ranges described herein. As non-

[0051] #14356316vl limiting examples, a dextran may have an acetyl substitution of between 1 and 3, and / or a succinyl substitution of between 0 and 2. In some cases, the sum of the substitutions is not greater than 3.

[0052] As a non-limiting example, in one embodiment, the dextran may have a structure: where n is a positive integer. In some cases, n can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, etc. In addition, in some cases, n may be no more than 200, no more than 190, no more than 180, no more than 170, no more than 160, no more than 150, no more than 140, no more than 130, no more than 120, no more than 110, no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 25, no more than 20, no more than 15, no more than 10, or no more than 5. Combinations of any of these ranges are also possible in some embodiments.

[0053] As another non-limiting example, the starch may have a structure: where n is a positive integer. In some cases, n can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, etc. In addition, in some cases, n may be no more than 200, no more than 190, no more than 180, no more than 170, no more than 160, no more than 150, no more than 140, no more than 130, no more than 120, no more than 110, no

[0054] #14356316vl more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 25, no more than 20, no more than 15, no more than 10, or no more than 5. Combinations of any of these ranges are also possible in some embodiments.

[0055] In some cases, the polymer may have a relatively low molecular weight. Without wishing to be bound by any theory, it is believed that lower molecular weights may improve the solubility of the polymer, e.g., as compared with higher molecular weights. For example, the molecular weight of the polymer may be at least 500 Da, at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 6 kDa, at least 7 kDa, at least 8 kDa, at least 9 kDa, at least 10 kDa, at least 11 kDa, at least 12 kDa, at least 13 kDa, at least 14 kDa, at least 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 45 kDa, at least 50 kDa, at least 55 kDa, at least 60 kDa, at least 65 kDa, at least 70 kDa, etc., and / or less than 70 kDa, less than 65 kDa, less than 60 kDa, less than 55 kDa, less than 50 kDa, less than 45 kDa, less than 40 kDa, less than 35 kDa, less than 30 kDa, less than 25 kDa, less than 20 kDa, less than 15 kDa, less than 14 kDa, less than 13 kDa, less than 12 kDa, less than 11 kDa, less than 10 kDa, less than 9 kDa, less than 8 kDa, less than 7 kDa, less than 6 kDa, less than 5 kDa, less than 4 kDa, less than 3 kDa, less than 1 kDa, less than 500 Da, etc. Combinations of these are also possible, e.g., a dextran may have a molecular weight of between 6 kDa and 10 kDa, between 5 kDa and 12 kDa, between 7 kDa and 9 kDa, or the like.

[0056] In addition, in some cases, the polymer may be cross-linked. Without wishing to be bound by any theory, it is believed that cross-linking of the polymer, e.g., physically, reversibly, or covalently, etc., may stabilize the shells around the gas, which may facilitate their administration to a subject. Once administered, the polymer may solubilize, e.g., due to the water solubility, which may then release the gas contained within the particle to the subject. In one set of embodiments, cross-linking of a polymer such as dextran may be achieved by exposing the dextran to an acidic environment, e.g., having a pH of less than 7, less than 6, less than 5, etc.

[0057] Without wishing to be bound by any theory, it is believed that at such pH’s, acid- induced protonation of carboxylic groups within the dextran, e.g., modified as discussed herein, may cause them to form cross-links. The soluble polymers can be induced in some cases to form stable shells or particles in aqueous solution under mixing (e.g., homogenization, sonication, etc.) upon addition of acids. In some cases, the mixing causes the polymer to absorb at a gasliquid interface, e.g., to form bubbles with unstable shells. In some cases, the addition of acid may cause the protonation of the carboxylic group of the

[0058] #14356316vl polymers and intermolecular crosslinking via hydrogen bond formation. This may, as a result, cross-link the polymers, thereby producing a shell encapsulating a gas core.

[0059] It should be understood that other polymers may be used instead of or in addition to dextran, in still other embodiments. For example, the polymer may be starch or other types of polysaccharides. Other non-limiting examples of suitable polymers that can be rendered amphiphilic or water-soluble include oligopeptides, poly(lactic-co-glycolic acid (PLGA), polyglutamic acid (PGA), hyaluronic acid, poly (citrate), poly(glycerol sebacate), chitosan, elastin, poly(carbonate), poly(hydroxy acids), poly anhydrides, polyorthoesters, polyamides, polycarbonates, poly alkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene, polyurethanes and co-polymers thereof, synthetic celluloses, polyacrylic acids, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone), ethylene vinyl acetate, various polyacrylate types, polynorbornene based polymers, various polyamides, copolymers and blends thereof. In addition, in certain embodiments, polymers such as these may be treated to become amphiphilic, water-soluble, and / or cross-linked, e.g., as described herein.

[0060] In some embodiments, the shell is free of one or more lipids. In addition, in some cases, the particle is formed from a single shell, e.g., surrounding a gas core. For example, the particle may not have a shell formed from a particle aggregate.

[0061] In some aspects, particles as described herein may be formulated as a pharmaceutical composition, for example, for administration or as a suspension (e.g., emulsions, foams, etc.) for storage, etc. Pharmaceutical compositions and suspensions of the particle may comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, viscosity enhancing agents (e.g., thickening agents), preservatives, solid binders, lubricants and the like, as suited to the particular formulation desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md., 2006) discloses various excipients used in formulating compositions and suspensions and known techniques for the preparation thereof. Except insofar as any conventional excipient is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the compositions or suspensions, its use is contemplated to be within the scope of this disclosure.

[0062] #14356316vl In some embodiments, the pharmaceutically acceptable excipient is at least 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, the excipient is approved for use in humans and for veterinary use. In some embodiments, the excipient is approved by United States Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0063] Pharmaceutically acceptable excipients used in the manufacture of the compositions and suspensions include, but are not limited to, inert diluents, dispersing agents, surface active agents and / or emulsifiers, disintegrating agents, preservatives, buffering agents, lubricating agents, and / or oils. Excipients such as coloring agents can be present in the compositions or suspensions, according to the judgment of the formulator.

[0064] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc. and combinations thereof.

[0065] Exemplary dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, crosslinked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.

[0066] Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate,

[0067] #14356316vl tartaric acid, and trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxy anisol (BHA), butylated hydroxy toluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus®, Phenonip®, methylparaben, Germall 115, Germaben II, NeoIone™, Kathon™, and Euxyl®. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative includes a chelating agent.

[0068] Exemplary buffering agents include, but are not limited to, dextrose buffer solutions (e.g., 10% dextrose buffer solutions), citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic saline, Ringer's solution, ethyl alcohol, etc. and combinations thereof.

[0069] For in vivo or medical applications, the compositions and suspensions as described herein may be generally isotonic or hypertonic with blood is in some embodiments. In some cases, the compositions and suspensions may also contain small amounts of one or more

[0070] #14356316vl isotonic agents. The isotonic agents may include physiological solutions commonly used in medicine and they comprise water, aqueous saline solution, e.g. 0.9% NaCl, 2.6% glycerol solution, lactated Ringer's solution, and 10% dextrose solution, biologically compatible organic solvents (e.g., DMSO), and / or commercially available intravenous fluid or blood.

[0071] The compositions and suspensions may also be mixed with volume expanders, such as Hextend, hetastarch, albumin, 6% Hydroxyethyl Starch in 0.9% Sodium Chloride Infusion (Voluven), etc. The compositions and suspensions can also be mixed with blood (e.g. packed red blood cells) or hemoglobin-based oxygen carriers. Additionally, the compositions and suspensions can be mixed in a physiologic buffer (e.g. tris(hydroxymethyl) aminomethane, “THAM”). This may be useful in a clinical situation of impaired ventilation. In other embodiments, the compositions and suspensions can contain one or more cryoprotectants, e.g., glycols such as ethylene glycol, propylene glycol, and glycerol. The compositions or suspensions may further comprise an aqueous solution comprises a calcium salt for enhanced stability.

[0072] The particles may also be suspended in a medium (e.g., an aqueous and / or organic medium), e.g., comprising a viscosity enhancing agent, according to some embodiments. Such particles may also be prepared in such a medium, as further described herein. Exemplary viscosity enhancing agents for use as a component of a storage medium and / or a preparative medium include, but are not limited to, com syrup (e.g., Clearsweet com symp (CS)); glycerin; cellulose derivatives (e.g., methylcellulose (MC); hydroxypropylmethylcellulose (HPMC); carboxymethylcellulose (CMC); microcrystalline cellulose (CC); ethyl cellulose; hydroxyethyl cellulose (HEC); hydroxypropyl cellulose (HPC); cellulose); gelatin; starch; hetastarch; poloxamers; pluronics; sodium CMC; sorbitol; acacia; povidone; carbopol; polycarbophil; chitosan; alginate; chitosan glutamate; hyaluronic acid; elastin; hyaluronan; maltodextrin DE; deoxy glycocholate (GDC); polymethacrylic acid; glycols (e.g., polymethylene glycol; polyethylene glycol); cyclodextrins (e.g., sulfobutylether B cyclodextrin); sodium tauro-dihydrofusidate (STDHF); and N-trimethyl chitosan chloride (TMC). In certain embodiments, the viscosity enhancing agent is corn symp (e.g., Clearsweet com syrup (CS)) or glycerin.

[0073] In certain embodiments, the particles are suspended in a medium (e.g., an aqueous and / or organic medium) comprises between about 5% to about 90% by weight of one or more viscosity enhancing agents, e.g., between about 5% to about 85%, between about 5% to about 80%, between about 5% to about 75%, between about 5% to about 70%, between about 5% to about 65%, between about 5% to about 60%, between about 5% to about 55%, between

[0074] #14356316vl about 5% to about 50%, between about 5% to about 45%, between about 5% to about 40%, between about 10% to about 80%, between about 15% to about 80%, between about 20% to about 80%, between about 25% to about 80%, between about 30% to about 80%, between about 35% to about 80%, between about 40% to about 80%, between about 45% to about 80%, between about 50% to about 80%, or between about 25% to about 75%, inclusive.

[0075] The medium (e.g., an aqueous medium and / or organic medium) which comprises one or more viscosity enhancing agents may be a viscous medium.

[0076] In certain embodiments, the gas-filled microparticle and / or nanoparticle compositions and suspensions described above can be formulated in a manner suitable for topical administration, e.g., as a liquid and semi-liquid preparation that can be absorbed by the skin. Examples of a liquid and semi-liquid preparation include, but are not limited to, topical solutions, liniments, lotions, creams, ointments, pastes, gels, and emulgels. In certain embodiments, the particle and / or pharmaceutical composition comprising the particle further includes a therapeutic agent, e.g., which can be, but are not limited to, hydrophilic or hydrophobic drugs, lipid-soluble drugs, nucleic acid-based drugs (including, e.g., genes, DNA, RNA, agRNA, smRNA, siRNAs, microRNAs, Crisper / Cas constructs, and / or nucleic acids for gene therapy), protein drugs such as antibodies, free radical scavengers, nitric oxide, a chemotherapeutic agent, a small molecule drug, and combinations thereof. In certain embodiments, the compositions and suspensions can be co-formulated with one or more additional therapeutic agents for co-delivery of the gas or gas mixture inside the microparticles and the one or more agents, which can be, but are not limited to, hydrophilic or hydrophobic drugs, lipid- soluble drugs, nucleic acid-based drugs such as siRNAs or microRNAs, protein drugs such as antibodies, or free radical scavengers. In certain embodiments, the therapeutic agent may be encapsulated in the core of the particle. In some embodiments, the particle comprises a therapeutic agent attached to the outer surface of the particle, e.g., by covalent attachment or by non-covalent association with the membrane.

[0077] Any of the particle-containing suspension described herein can be in suspension form or in dry powder form (e.g., obtained via spray drying or by lyophilization). When in dry powder form, the suspension can, in some cases, be mixed with a solution such as saline immediately before use.

[0078] The gas-filled particle compositions or suspensions described above can be used for gas delivery shortly after their preparation. In some embodiments, they can be stored under suitable conditions (e.g., refrigerated conditions) before administration.

[0079] #14356316vl Further contemplated in certain embodiments are kits or pharmaceutical packs comprising a particle and instructions for use. In certain embodiments, the kit comprises a container housing a particle, a container housing a pressurized aqueous phase mixture, and instructions for mixing the particle and the aqueous phase. In certain embodiments, the container housing the particle and the container housing the aqueous phase are separate compartments within a single container.

[0080] Another aspect is generally drawn to systems and methods for producing particles such as those described herein. In some cases, bubbles of gas may be formed in a solution comprising dextran or other polymers (e.g., as described herein), and the solution may be treated to acidify or reduce its pH to cause the dextran or other polymers to cross-link and absorb onto the surface of the bubbles, thereby forming particles having a shell surrounding a gas. The bubbles of gas may include any of the gases described herein (for example, air, oxygen, etc.), and the solution may contain any of the polymers described herein, for example, water-soluble polymers that can be cross-linked, e.g., upon exposure to a pH of less than 7. After absorption, in some embodiments, the solution can be removed, for example, and replaced with another solution, e.g., to form a pharmaceutical composition.

[0081] The bubbles of gas may have the same or different sizes, and may include any of the sizes described herein. For example, the bubbles may have an average diameter of less than 500 micrometers, less than 300 micrometers, less than 200 micrometers, less than 100 micrometers, less than 50 micrometers, less than 30 micrometers, less than 20 micrometers, less than 15 micrometers, less than 10 micrometers, less than 5 micrometers, less than 3 micrometers, less than 2 micrometers, less than 1 micrometer, less than 0.5 micrometers, less than 0.3 micrometers, less than 0.2 micrometers, less than 0.1 micrometers, etc. In some cases, the bubbles may have an average diameter of at least 0.1 micrometers, at least 0.2 micrometers, at least 0.3 micrometers, at least 0.5 micrometers, at least 1 micrometer, at least 2 micrometers, at least 3 micrometers, at least 5 micrometers, at least 10 micrometers, at least 15 micrometers, at least 20 micrometers, at least 30 micrometers, at least 50 micrometers, at least 100 micrometers, at least 200 micrometers, at least 300 micrometers, at least 500 micrometers, etc. Combinations of any of these are also possible. For example, the bubbles may have an average diameter of between 5 micrometers and 10 micrometers, between 1 micrometer and 5 micrometers, between 0.5 micrometers and 2 micrometers, between 0.5 micrometers and 3 micrometers, between 0.2 micrometers and 2 micrometers, between 3 micrometers and 15 micrometers, etc.

[0082] #14356316vl In some cases, the solution may be slowly acidified by the introduction of an acid, e.g., HC1, H2SO4, acetic acid, etc. Other methods of cross-linking include, but are not limited to, ions (e.g., calcium), light, temperatures, small molecule chelating agents, polymeric complexing agents, or the like. In some cases, this may be performed until a certain thickness of polymer has been absorbed onto the bubbles. For example, the reaction may occur until the thickness of the polymer surrounding the bubble is at least 1 nm, at least 2 nm, at least 3 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 500 nm, etc. Without wishing to be bound by any theory, it is believed that the amphiphilic character of the polymer may promote its absorption on the bubbles, e.g., at the gas / liquid interface.

[0083] In yet another aspect, provided are methods of delivering or administering a gas to a subject in need thereof. In some cases, a pharmaceutical composition comprising particles having a shell surrounding a gas core, such as those discussed herein, may be administered to a subject. In some cases, the composition may also comprise a pharmaceutically acceptable excipient, e.g., as discussed herein. The subject may be human, or a non-human animal. Examples of subjects include, but are not limited to, a mammal such as a cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, cat, a primate (e.g., a monkey, a chimpanzee, etc.), or the like. In some cases, the subject is a non-mammal, such as a bird, an amphibian, or a fish.

[0084] The compositions containing particle suspensions may be administered locally or systemically, depending on the condition to be treated, in certain aspects. The compositions may be administered via injection. In some embodiments the compositions can be administered as continuous infusions. In certain embodiments, the pharmaceutical composition is administered to the subject by intravenous, intraosseous, intraperitoneal, intraarterial, subcutaneous, and / or intramuscular injection or infusion. In certain embodiments, the pharmaceutical composition is administered to the subject topically, orally, enterally, sublingually, intranasally, or by inhalation. In certain embodiments, topical delivery is delivery to pleural, skin, peritoneum, or facial. In some cases, the compositions are administered directly to the tissue or organ in need of treatment. In certain embodiments, the pharmaceutical composition is administered to the subject by inhalation or nebulization. In certain embodiments, the pharmaceutical composition is administered topically to the skin, e.g., to a wound or lesion. In some embodiments, the particles can be administered inhalationally (e.g., in an asthma attack), topically (to the pleural or peritoneal cavity, to the skin, to a burn, to a wound, to the fascia, to the muscles, to the intestines or other organs), enterally (orally, sublingually, enterally, rectally, etc.).

[0085] #14356316vl In certain embodiments, the subject is or is suspected of experiencing local or systemic hypoxia. In certain embodiments, the subject has or is suspected of having a disease or disorder selected from the group consisting of congenital physical or physiologic disease, transient ischemic attack, stroke, acute trauma, cardiac arrest, exposure to a toxic agent (e.g., such as carbon monoxide), heart disease, hemorrhagic shock, pulmonary disease, acute respiratory distress syndrome, infection (e.g. sepsis), acute decompression sickness, and multi-organ dysfunction syndrome. In some embodiments, the pharmaceutical composition may be delivered to a solid tumor that is hypoxic in one or more regions of the tumor. In some embodiments, the pharmaceutical composition may be administered in combination with radiotherapy, radiation therapy, cancer immunotherapy, or any combinations thereof.

[0086] Particles such as any of those described herein can be used in various medical or nonmedical applications, e.g., but not limited to therapeutic applications in which the stable particles are used to deliver a gas to a subject in need thereof; as a contrast agent in diagnostic imaging (e.g., ultrasound imaging, Plasmon- surface enhanced imaging, or MRI imaging); as additives in cosmetic and / or personal care compositions, e.g., for viscosity enhancement and color modulation; as additives in food products and / or beverages, e.g., to improve texture and / or stability; as antimicrobial or pesticides (e.g., for plants or crops, or for water treatment, or for treatment of skin diseases); as fuel additives to improve fuel efficiency; drinking water treatment or treatment for ocean non-salt water (e.g., an algicide); etc. Additional applications include, but are not limited to use of the stable particles described herein to form acoustic barriers; addition of the stable particles described herein in a flooding fluid for enhanced oil delivery; and addition of the stable particles described herein in a hydrogel tissue scaffold as porogens or to facilitate gas transport.

[0087] Accordingly, some aspects provided are methods of delivering a gas to a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a particle as described herein, e.g., with a pharmaceutically acceptable excipient. The gas-filled particles described herein can be used to deliver a gas into a subject, thereby treating various diseases and conditions. The gas-filled particles may be administered to any subject, tissue or organ in need thereof, i.e., in need of the gas to be delivered, e.g., by intravenous, intraosseous, intraperitoneal, intraarterial, subcutaneous, and / or intramuscular injection or infusion; alternatively it can be topically applied as a powder or wetted, or inhaled, ingested or applied topically to a body cavity, such as the pleura, the pericardium or the peritoneum or administered peritoneal or retroperitoneal. The particles may be administered alone or in combination with other treatments as an adjunctive

[0088] #14356316vl therapy. Depending upon the need of a subject, the particle can be designed such that they release the gas or gas mixture immediately following administration (e.g., <10 milliseconds to 1 minute). In some cases, the particles can be designed to provide sustained release of the gas or gas mixture, and / or to persist in vivo until they reach the target tissue, where the membrane collapses to release the gas or gas mixture.

[0089] The term “treating” as used herein refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease / disorder, or a predisposition toward the disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease / disorder, the symptoms of the disease / disorder, or the predisposition toward the disease / disorder.

[0090] In certain embodiments, the subject is or is suspected of experiencing local or systemic hypoxia. In certain embodiments, the subject has or is suspected of having a disease or disorder selected from the group consisting of congenital physical or physiologic disease, transient ischemic attack, stroke, acute trauma, cardiac arrest, exposure to a toxic agent, heart disease, hemorrhagic shock, pulmonary disease, acute respiratory distress syndrome, infection, solid hypoxic tumor, and multi-organ dysfunction syndrome.

[0091] An “effective amount” is the amount of the particles that alone, or together with one or more additional therapeutic agents, produces the desired response, e.g. increase in the local or systemic level of a desired gas such as oxygen in a subject or increases the tissue PO2 in a particular target organ. In the case of treating a particular disease or condition, the desired response can be inhibiting the progression of the disease / condition. This may involve only slowing the progression of the disease / condition temporarily, although more preferably, it involves halting the progression of the disease / condition permanently. This can be monitored by routine methods. The desired response to treatment of the disease or condition also can be delaying the onset or even reducing the risk of the onset of the disease or condition. An effective amount will depend, of course, on the particular disease / condition being treated, the severity of the disease / condition, the size of the patient, the volume of distribution of the drug, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of a health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a moderate dose of the particles be used, that is, the highest safe dose according to sound

[0092] #14356316vl medical judgment, taking into account that following a hypoxic injury, for example, an excessive or even normal oxygen tension may be harmful during the recovery period.

[0093] Suspensions containing oxygen-filled particles as described herein can be used to restore the oxygen level in a patient experiencing or being suspected of experiencing local or systemic hypoxia via any of the methods described herein, in accordance with certain embodiments. Thus, they have broad therapeutic utilities, including treatment of traumatic brain injury, cardiac arrest (via either intraarterial infusion or intravenous infusions), promotion of wound healing, topical augmentation of oxygen delivery (as topically administered to a body cavity or enterally administered) and preservation of organs during transplant, etc.

[0094] The particles may be administered in an effective amount and at suitable rate for increasing or maintaining the PO2 in a subject following administration. Typically, the particles are administered in an effective amount and at suitable rate to deliver an effective amount of oxygen to a subject to ischemic tissues or to desaturated blood in a time ranging from 0.5 to 30 seconds following administration, wherein the amount of oxygen that is delivered is effective to restore PO2 levels to normal levels or prevent or alleviate hypoxic injury. In certain embodiments, the particles provide sustained release of oxygen; such particles may be used, for example, to deliver oxygen or other gas to the brain and other tissues. In certain embodiments, the particles can deliver supers aturation of oxygen, e.g., for hyperbaric therapy.

[0095] Some embodiments are generally directed to oxygen supplementation via the enteral route. In some cases, oxygen-filled particles and, optionally, lipid nutrients, carbohydrates, or other nutrients found in blood (e.g., glucose and other blood components), can be delivered via the enteral route, e.g., to a site in the abdominal cavity, such as the intestine or the peritoneum, to provide an alternate source of intestinal oxygenation and prevents or mitigates intestinal ischemia, which may contribute to necrotizing enterocolitis, a leading cause of pediatric morbidity and mortality in preterm infants. This may also decrease the burden of anaerobic bacteria in the bowel, decreasing the risk of bacterial translocation and sepsis. This can also benefit prematurely born infants as it may decrease toxicity to premature lungs, prevents retinopathy of prematurity, and also provides lipid nutrition at the same time. In addition, it may be used in adults such as COPD patients, who require supplemental oxygen for some reason. It may also provide an alternative method of providing supplemental oxygen to critically ill patients such as ARDS patients, in whom increasing oxygen delivery through the lungs may be prohibitively injurious.

[0096] #14356316vl Certain embodiments are generally directed to the preservation of organ and / or blood, e.g., in vitro. Low blood oxygen tensions within stored blood may contribute to the blood storage defect, causing cells within the plasma to generate lactate and toxins, which may decrease the therapeutic value of transfused blood and diminish its shelf life. Oxygen-filled particles may be added to a blood sample periodically to prolong in vitro blood storage. In an explanted organ, a suspension containing oxygen-filled particles can be delivered into a blood vessel in an organ to provide oxygen supply, thereby ameliorating tissue damage due to hypoxia. This may be useful in preserving organs to be used in transplantation. In addition, oxy gen-filled particles can be added to a blood sample periodically to prolong in vitro blood storage in some embodiments.

[0097] Int. Pat. Apl. Pub. Nos. WO 2009 / 043031, WO 2012 / 065060, WO 2013 / 151682, WO 2014 / 144364, WO 2014 / 143808, and WO 2018 / 160752 are each incorporated herein by reference in their entireties. In addition, U.S. Pat. Apl. Ser. No. 63 / 706,473 is incorporated herein by reference in its entirety.

[0098] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0099] EXAMPLE 1

[0100] Acute respiratory failure can cause profound hypoxemia that leads to organ injury or death within minutes. When conventional interventions are ineffective, the intravenous administration of oxygen can rescue patients from severe hypoxemia, yet at the risk of microvascular obstruction and of toxicity of the carrier material. This example describes polymeric microbubbles as carriers of high volumes of oxygen (350-500 mL of oxygen per litre of foam) that are stable in storage yet quickly dissolve following intravenous injection, reverting to their soluble and excretable molecular constituents. In swine with profound hypoxemia owing to acute and temporary (12 min) upper-airway obstruction, the microbubble-mediated delivery of oxygen led to the maintenance of critical oxygenation, to lowered burdens of cardiac arrest, to improved survival, and to substantially improved neurologic and kidney functions in the surviving animals. These examples underscore the importance of maintaining a threshold of oxygenation and the promise of injectable oxygen as a viable therapy in acute and temporary hypoxemic crises.

[0101] This example shows intravenous injection of oxygen via polymeric microbubbles that are stable in storage yet quickly dissolve following intravenous injection led to the maintenance of critical oxygenation and to improved survival in swine with profound hypoxemia.

[0102] #14356316vl In particular, this example shows a rational approach to design polymeric microbubbles (PMBs) as gas carriers for translating IVO2. PMBs were designed in this example to instantaneously dissolve in physiologic media absent a diffusion sink, their shells reverting to soluble, excretable molecular constituents, thus allowing rapid delivery of high volumes of oxygen without observable acute safety or toxicity risks. It is also shown that injections of oxygen via PMBs substantially improve survival and neurologic outcomes in a realistic animal model of severe hypoxemic cardiac arrest. These findings illustrate the therapeutic importance of maintaining a critical threshold of oxygenation during acute, profound hypoxemic events, highlighting the unique potential of IVO2 as a viable therapy.

[0103] Gas carrier design. Several factors were considered in the general design of a pharmaceutically acceptable gas carrier. First, the carriers should rapidly release oxygen upon contact with blood. Second, any particulate that is injected should rapidly dissolve following administration to avoid vascular obstruction. Third, carrier fluids should be minimized to avoid fluid overload. Fourth, the carrier materials should have low toxicity and be efficiently cleared to minimize long-term side effects.

[0104] This example demonstrates a broadly applicable method of pH-induced interfacial cross-linking to create stable, pH-responsive PMBs using low MW and water-soluble polymers in aqueous solution without the use of an organic cosolvent. At blood pH, PMB shells revert into their molecularly soluble components (Fig. 1A), an FDA-endorsed strategy to minimize the safety risks of injectable nanomaterials. To demonstrate this approach, a low MW dextran (6 kDa) was first selected as a starting material and chemically modified with acetyl and carboxyl moieties (LmD) to create a pH-responsive amphiphile that is soluble at physiologic pH but insoluble in an acidic environment (Fig. 1A). To prepare PMBs, the LmD polymer was dissolved in a pH-adjusted dextrose solution (pH 6.5) and homogenized at the air-water (a / w) interface, while the mixture was slowly acidified using dilute hydrochloric acid (target pH ~3.5). Homogenization created bubble templates to promote absorption of LmD at the a / w interface due to its amphiphilic character, while LmD was cross-linked via the acid-induced protonation of carboxylic groups. This process resulted in the formation of a thin shell (<50 nm) MB around the gas core (Fig. IB). Interfacial cross-linking was driven by intermolecular hydrogen bonding and other Van der Waals interactions. The IR absorption of C=O of the carboxylic acid groups in polymer shells (Fig. 1C) revealed that a majority of them were in bonded states (1727 cm'1, 1734 cm'1) along with a smaller fraction of unbonded ones (1740 cm'1). The yield of PMBs significantly increased with dextrose concentration (Fig. ID), likely due to its promotion of polymer aggregation and greater

[0105] #14356316vl density at the a / w interface at lower pHs. The PMB shell revealed a hydrogel-like property and osmotic balance with the surrounding fluid; washing PMBs manufactured in 30% dextrose with pure water caused swelling of the shell and water influx with replacement of the gas core (Fig. IE); nonetheless, the sterilely fabricated PMBs with 30% dextrose can be washed and stored in 10% dextrose (DIO), a standard clinical IV fluid. Packing density (and therefore gas carrying capacity) of the foam was optimized by varying homogenization speed, with an optimal gas fraction of 62 + / - 2% (vol gas / vol foam) and a mean particle diameter ~ 5 micrometers (Figs. IF and 1G). The thin shells of PMBs were highly gas permeable, allowing air- filled PMBs (aPMBs) to be readily converted to oxy gen-filled PMBs (oPMBs) through passive purging of the headspace with oxygen. oPMBs can be stored in a closed container at room temperature and are stable for months (Fig. 1H). The long-term stability of oPMBs was evaluated via an accelerated stability test at various temperatures, oPMBs stored in glass syringes were stable up to 45 °C for up to 30 days without changes in foam volume or size distribution, while loss of oPMBs were observed at 60 °C. Based on these results, the shelf-life of oPMBs was expected to be at least 6 months at room temperature and at least 1 year under refrigerated conditions. The sterility of each manufactured lot was maintained and monitored prior to in vivo use. To show the material tunability of this system, the application of this approach to prepare PMBs from a low MW carboxylated hydroxyethyl starch polymer was also demonstrated.

[0106] Fig. 1 shows the design and fabrication of LmD PMB gas carriers for IVO2 therapy. Fig. 1A shows that LmD PMBs were manufactured through a process of homogenization and simultaneous titration of acid for interfacial cross-linking of the LmD polymer, which was solidified through hydrogen bonding. Eollowing contact with blood, the PMBs rapidly dissolved to deliver gas and their shells reverted into their low MW and soluble molecular constituents, which were excreted via urine and hepatic clearance. Pig. IB shows cryo-SEM imaging of LmD PMBs, depicting their thin shell and smooth surface. Pig. 1C shows that the IR absorption peak of carbonyl groups (black curve) in LmD PMBs indicated they existed in various H-bound states (deconvoluted Gaussian peaks by taking second derivative). Pig. ID shows that light microscopy of PMB solutions homogenized in varying dextrose concentrations illustrated that PMB concentration (i.e., yield) increased with increasing dextrose additives. Scale bar 10 micrometers. Pig. IE shows the effect of osmolarity of carrier fluids on LmD PMBs that were fabricated under 30% dextrose. LmD PMBs originally made with 30% dextrose were not stable in water but were stable in D10 and solutions with higher dextrose concentrations. Data are mean + / - SD, biological replicates. Pig. IP shows

[0107] #14356316vl size distributions and microscopy of LmD PMBs fabricated under various homogenization speed under 30% dextrose. Scale bar 10 micrometers. Fig. 1G shows the gas carrying capacity of LmD PMBs foams fabricated under various speeds. Data are means + / - SD, biological replicates. Fig. 1H shows that the sterilely fabricated LmD PMBs stored in DIO at room temperature did not change size distribution after 3 months.

[0108] EXAMPLE 2

[0109] Dissolution of PMBs ex vivo. The intravenous administration of a gas may require the rapid dissolution of gas carriers to avoid vascular obstruction. Subsequently, the carrier materials may be biocompatible and rapidly cleared. As noted earlier, the LmD polymer itself exhibits pH-responsive behavior in solution due to the presence of carboxylic acid groups (pKa ~ 4.8) and is molecularly soluble above pH 5 (Fig. 2A). In this example, the mechanism of dissolution of PMBs following injection hinged upon the pH-based deprotonation of carboxylic groups, which increases the solubility and hydration of polymers that compose the shell. This caused water influx in the shell, increasing surface tension and destabilizing the gas core, promoting its dissolution. The deprotonated shell simultaneously reverted to small and soluble components. Notably, unlike lipid coated bubbles, which require a gas concentration gradient (i.e., sink) to dissolve, PMBs dissolve at a physiologic pH within seconds even in the absence of a sink. To examine whether PMB shells fully dissolve and revert to soluble LmD constituents, in tis example, PMBs were added into phosphate buffered saline (PBS) solution of varying pH under stirring and DLS size measurements were confirmed 2 minutes following admixture (earlier timepoint was not obtained by DLS due to the sampling limitations of the instrument). Between pH 9.0 and 5.0, PMB shells were all fully dissolved within 2 minutes, reverting to soluble polymers of similar sizes of LmD solutions prepared from solid states (Fig. 2A). Solubilized LmD polymer had a mean hydrodynamic radius less than 10 nm and an estimated MW ~12 kDa (determined by NMR), well below the MW cutoff for glomerular filtration (30 - 45 kDa). In contrast, dissolution of previous IFNP MBs, which used hydrophobic polymers of higher MW (>60 kDa), reverted into large and insoluble nanoparticles (>100 nm) that visibly precipitate over time.

[0110] To better investigate the pH-dependent dissolution kinetics, PMBs mixed with PBS while continuously applying ultrasound were examined. Like various polymeric shelled microbubbles, the gas core of PMBs creates acoustic backscatter and produces contrast in proportion to the presence of gas bubbles within the field of view. The decrement in contrast intensity (i.e., bubble dissolution) was shown to be pH-dependent: at pH 9.0, 7.2, and 6.5, PMBs were no longer visible within 2 to 3 seconds, while dissolution of the gas core was

[0111] #14356316vl prolonged at pH<6 (Figs. 2B, 2C). (To note, although the LmD shell is less soluble at pHs 4.8 and 3.8, it was noticed the gas core of PMBs slowly became fluid- filled as shown by the slow decrease in echo intensity; this may be because the salts in PBS affected the swelling of the hydrogel-like shells). While it is known that ultrasound may contribute to loss of MBs due to inertial cavitation, Fig. 2B suggested that pH is the dominant factor affecting dissolution rate. To further account for acoustic destruction, the same experiments were performed while applying ultrasound only at selected terminal time points, finding similar dissolution rates (Figs. 2D, 2E).

[0112] To further examine dissolution of the shell (separate from that of the gas core), UV- Vis spectroscopy was performed. In this construct, it was expected that an increase in absorbance from baseline could be caused by either undissolved gas cores or large polymeric aggregates (i.e. undissolved shell or aggregated constituents). From pH 9.0 to 6.0, UV absorbance reached baseline within 2-3 seconds, similar to the kinetics in acoustic studies, indicating both that the gas core had dissolved, and the shell reverted to its soluble constituents in that time (Figs. 2F, 2G). Between pH 5.5 and 5.0, the return to baseline was much longer than in the acoustic study, suggesting that within this pH range the gas core dissolves first, and the shell required more time to revert to soluble polymers. These findings were consistent with the pH-triggered dissolution mechanism of PMBs that is essentially an acid-base reaction, the rate of which is proportional to the concentration of hydroxyl ions in the solution and limited by diffusion. This mechanism also explained some discrepancies of dissolution kinetics seen at lower pHs. For example, in contrast to UV-Vis, DLS showed PMBs were fully dissolved at 2 minutes at pH 5.5 and 5.0, this may be due to lack of sufficient mixing in Uv-vis experiments. However, at pH>6.0, the dissolution kinetics measured from various methods were all in good agreement. Collectively, these results validate this design for the new gas carrier and established that both the gas core and the shell of PMBs rapidly dissolve at pH levels (7.5 to 6.5) that are relevant to intravenous injection, as the blood pH rarely drops below 6.5 even in extreme instances.

[0113] Fig. 2 shows LmD PMBs rapidly dissolve at physiologic pH. Fig. 2A shows DLS measurement of size following mixing of LmD PMBs mixed in PBS solution for 2 minutes at varying pH. LmD PMBs fully dissolve above pH 5 and revert to their soluble components with a mean size < 10 nm, similar to those of LmD solutions prepared from solid states. In contrast, the previous generation of IFNP MBs (made from more hydrophobic polymers) led to formation of much larger nanoparticles. Fig. 2B shows phantom sonography of aPMBs in aerated PBS shows the pH-dependent dissolution rate, evidenced by the disappearance of

[0114] #14356316vl contrast intensity produced by the gas core under continuous ultrasound. In the absence of a gas sink, PMBs rapidly dissolved above pH 6 within seconds. Data (means + / - SEM) presented as changes in contrast / bright area from baseline. (To note, although the EmD shell is less soluble at pHs 4.8 and 3.8, it was noticed the gas core of PMBs slowly becomes fluid- filled as shown by the slow decrease in echo intensity; this may be because the salts in PBS affected the swelling of the hydrogel-like shells). Fig. 2C shows representative images from phantom sonography study that show the dissolution profile of aPMBs at different time points at various pHs. Figs. 2D and 2E show that to account for any destructive effect that ultrasound itself has on PMBs, the experiment was repeated while only applying ultrasound at the expected dissolution time from Fig. 2B, showing similar dissolution times even absent the application of continuous ultrasound. Data (means + / - SEM) presented as change in area of contrast / brightness from baseline, analyzed by student’s t-test. Fig. 2F and 2G shows the dissolution of the shell and gas core was then studied using UV-vis absorbance spectroscopy. Similar to the characterization using ultrasound (which detects only dissolution of the gas core), UV-vis returns to baseline within seconds at pH above 6, suggesting both that the gas core had dissolved and that the shell has broken down into its constituent components. At more acidic pH, return of UV-Vis absorbance to baseline took 10 minutes or longer.

[0115] Contrasting this with sonographic experiments in Fig. 2B in which ultrasound scatter returned to baseline within 90-260 seconds, these findings suggest that following dissolution of gas core, the remaining shell constituents take additional time to dissolve and revert to soluble components. All repeated measurements are biological replicates.

[0116] EXAMPLE 3

[0117] Effects of PMBs on acute hemodynamics. Prior injectable gas carriers that did not exhibit a triggered dissolution mechanism caused pulmonary vascular obstruction due to bubble persistence following injection (Fig. 3 A, 3B). Any intravenously injected fluid would immediately travel from the injection site to the right atrium and ventricle, from which it is then ejected into the pulmonary circulation prior to returning to the left heart to enter the arterial system. The pulmonary capillaries are the smallest blood vessels thus have the highest susceptibility to occlusion; if particulate matter or gas embolism from gas carriers obstruct pulmonary capillaries, pulmonary vascular resistance could (PVR) increase. To provoke vascular obstruction in this model, air- filled aPMBs were used rather than oPMBs, since the driving gradient for oxygen egress exceeds that of nitrogen due to the high concentration of deoxyhemoglobin in the venous system. Following a baseline period, rats in the test group (n = 4, weight 503+ / -52 g) received 5 repeated injections of 5 ml of 50% vol gas / vol foam in

[0118] #14356316vl DIO (-2.5 ml gas per injection) every 3 minutes (total 12.5 ml of air) followed by a 60- minute observation period; the control group (n=5, 518+ / -40 g) received an equal volume of DIO. The injected gas content was ~1.7 mL / kg / minute, representing the equivalent of 50- 100% of basal oxygen consumption of a human. In both groups (Figs. 3C, 3D), PVR decreased during the injection period relative to baseline, and there were no differences between groups (-64.5+ / -40.1 mmHg / (mL / kg / min) PMB vs -45.2+ / - 27.2 mmHg / (mL / kg / min) control, P=0.41), likely representing preload recruitable stroke work and increased cardiac index in both groups. Relatedly, mean arterial blood pressure increased similarly in both groups during the injection period (Figs. 3E, 3F) (53.0+ / -20.6% PMB vs 57.6+ / -21.7% above baseline, P=0.69). Taken together, this acute hemodynamic profile suggests an absence of pulmonary obstruction following serial, rapid PMB injections, and is distinct from the profile seen with rapid injection of LOMs and gas carriers composed of PLGA.

[0119] To verify the dissolution of PMBs in vivo, transthoracic echocardiography following injection of PMBs was performed and compared with that of LOMs (Fig. 3G). aPMBs or oPMBs (70% foam) were continuously infused at various rates up to 12 mL gas / kg / min; oxygen-filled LOMs (50% foam, equal gas volume) were infused only at the lowest rate (4 mL gas / kg / min), as higher rates caused hemodynamic collapse. Injection of LOMs immediately opacified all four heart chambers (Fig. 3H), demonstrating trans-pulmonary passage of undissolved LOMs. In contrast, aPMBs and oPMBs were not visible in the LV even when injected at a 3X higher rate (Fig. 3L3L). Compared with LOMs, opacification of the right ventricle was also less pronounced following injection of aPMBs and oPMBs injected at an equivalent rate, supporting evidence for their rapid in vivo dissolution. Right heart opacification disappeared within seconds following the end of PMB injections, whereas circulating LOMs were still visible 10 minutes after injection, highlighting their in vivo persistence. Taken together, these data suggested that PMBs dissolve rapidly following even rapid injection and did not cause vascular obstruction even in the absence of a sink, while delivering a high gas payload.

[0120] Fig. 3 shows that the pH-triggered, rapid dissolution mechanism of LmD PMBs avoids vascular obstruction and hemodynamic instability. Fig. 3A shows that the rapid dissolution of PMBs is important to their in vivo safety. Previously described gas carriers did not dissolve rapidly or coalesced following injection, leading to pulmonary vascular obstruction. Fig. 3B shows that in contrast, PMBs dissolved so rapidly following injection that they existed mainly as soluble molecular constituents by their first contact with the pulmonary circulation. Figs. 3C-3F show a hemodynamic safety study. n=4 for treatment,

[0121] #14356316vl n=5 for DIO control. Continuous measurements collected as biological replicates. Pulmonary vascular resistance (PVR) (Fig. 3C) was not significantly different from baseline following 5 injections of 5 ml 80% aPMBs (50% vol air / vol) over 1 minute each. Data (means + / - SEM) presented as mean percent change from baseline. Mean PVR during each experimental period (Fig. 3D) did not change during or following infusions of aPMBs during either the injection or observation period. Data are means + / - SEM, P values calculated by two-way ANOVA. Mean arterial blood pressure (MABP) (Fig. 3E) increased during injection of PMBs and returned to baseline thereafter. Data presented as mean percent change from baseline, error = SEM. Mean MABP during each experimental period (Fig. 3F) did not change during or following infusions of aPMBs during either the injection or observation period. Data are means + / - SEM, P values calculated by two-way ANOVA. (Figs. 3G-3J). Representative transthoracic echocardiography images during infusion of PMBs and LOMs through the left parasternal window for a four-chamber view. Fig. 3G shows a control animal, four chamber view with right atrium (RA), left atrium (LA), right ventricle (RV) and left ventricle (LV). Fig. 3H shows high opacification in both left and right ventricles of an animal injected with lipid oxygen microparticles (LOMs) at a rate of 4 mL / kg / min. Fig. 31 shows moderate opacification in the right ventricle of an animal injected with intravenous oxygen (IVO2) of oPMBs at a rate of 4 mL / kg / min, no visible signal noted in the left chambers. Fig. 3 J shows moderate opacification in the right ventricle of an animal injected with intravenous aPMBs (IV Air) at a rate of 4 mL / kg / min, no visible signal noted in the left chambers. Figs. 3K and 3L show the percentage of opacified areas in the right (Fig. 3K) and left (Fig. 3L) ventricles relative to respective ventricle area were quantified during administration of IVO2, IV AIR and LOM at the flow rates shown, each during a 1 -minute infusion. Data are means + / - SEM, comparisons by one-way ANOVA, Tukey’s multiple comparison test.

[0122] EXAMPLE 4

[0123] Efficacy of PMBs in hypoxemia-related cardiac arrest in swine. Having demonstrated the acute safety of PMBs, this example assessed their effect on a clinically realistic, extreme model of hypoxemic respiratory failure and in-hospital cardiac arrest (IHCA) (Fig. 4A). Briefly, Yorkshire swine were anesthetized and instrumented, including tracheal intubation and placement of arterial and venous catheters. Following a period of observation under IV sedation and neuromuscular blockade while breathing 21% oxygen, the swine underwent 12 minutes of apnea / asphyxia. Animals experiencing cardiac arrest (defined as systolic blood pressure <40 mmHg for 5 seconds or longer) received high-quality, chest compression CPR and rhythm-directed resuscitative interventions, including medications and defibrillation

[0124] #14356316vl according to current standards. At minutes 6, 8 and 10 of injury, swine were randomized to receive either IVO2 (combined total 400 ml of 35% vol 02 / vol foam oPMBs, containing -140 ml oxygen, n=8) or an equal volume of oxygenated DIO (n=10). Given that the mean measured resting oxygen consumption (VO2) during the baseline period in this experiment was 73.2 mL / min, this volume represents the provision of -30% of resting oxygen consumption for the last 6 minutes of asphyxia. After 12 minutes, the airway was opened, and ventilation restored. CPR was continued for up to 30 minutes or until the return of spontaneous circulation (ROSC). Surviving animals were then maintained for 4 days in an ICU environment, including mechanical ventilation, extubation readiness testing, inotropic support, and seizure monitoring and treatment according to a standardized protocol. On day 4, brain magnetic resonance imaging (MRI) was performed, followed by euthanasia and pathological analysis.

[0125] Effect of oPMBs on resuscitation metrics. There were no differences in baseline characteristics between groups, including age (43 days oPMB vs 49 days control, P=0.08) or weight (12.1+ / - 1.1 vs 12.0+ / - 1.3 kg, P=0.953). At 6 minutes of asphyxia / apnea, a similar number of animals experienced cardiac arrest. CPR quality was excellent in both groups with no significant differences in compression rate, compression fraction, or compression depth. Arterial oxygen saturation (SaCE, measured by co-oximetry on blood gas every odd minute) reached undetectable levels (<3%) at 6 minutes, and was significantly higher in IVCh-treated swine than in those receiving control at 9 minutes (24+ / - 14% vs 4+ / -2%, P=0.012), 11 minutes (23+ / -7% vs 3+ / -l%, P=0.017) and 13 minutes (96+ / -8% vs 77+ / -36%, P=0.024, Fig. 4B). Similarly, the partial pressure of oxygen in arterial blood (PaO2) was significantly higher in IVO2-treated swine at 7, 9 and 11 minutes. Arterial carbon dioxide tension was higher at 7, 9, 11, and 13 minutes in IVO2-treated swine (P<0.05, Fig. 4C), presumably due to preserved cellular metabolism and CO2 production (though this may be partially explained by the Haldane effect as well). Although all swine experienced cardiac arrest, IVO2 restored the circulation during asphyxia in a number IVO2-treated swine (Fig. 4D), such that the duration of CPR (Fig. 4E) and resuscitative doses of epinephrine indicated in the protocol (Fig. 4F) were significantly lower in IVO2-treated swine; IVO2-treated swine also had significant improvements in mean arterial blood pressure during the treatment period (Fig. 4G). Following relief of airway obstruction, swine in the IVO2-treated group were more likely to achieve the return of spontaneous circulation (ROSC) (100% vs 30%, P=0.003) and overall survival (88% vs 30%, P = 0.007) (Fig. 4H).

[0126] #14356316vl Effect of oPMBs on organ injury. Only 3 out of 10 swine in the control group achieved ROSC, all of which experienced severe neurologic injury: none extubated successfully, and all experienced refractory status epilepticus and diabetes insipidus (a phenomenon indicative of profound brain injury). Of the 8 achieving ROSC in the IVO2- treated group, 5 were successfully extubated within 24 hours and were able to ambulate and eat and drink independently by 3 days post-injury. Even amongst only surviving swine, Swine Neurologic Deficit Scores were significantly lower at post- injury days 1-3 in IVO2 treated swine (Fig. 41). Glial fibrillatory acidic protein (GFAP), a brain astrocytic protein released in proportion to brain cellular injury, was nearly two orders of magnitude higher at 4 days in surviving control swine than in those receiving IVO2 (0.3+ / -0.2 IVO2 vs 21.8+ / - 12.4 ng / ml controls, P<0.001) (Fig. 4J). None of the 3 surviving control animals exhibited any detectable intracranial blood flow by magnetic resonance angiography, and all had evidence of a generalized edema and uncal and tonsillar herniation (Fig. 4K). When assessed using a manual segmentation of the diffusion imaging, IVO2-treated swine exhibited significantly lower volumes of white matter injury (2,269 [IQR 1,560-3,533] mm3) compared with controls (20,784 [19,154 to 23,556] mm3, P < 0.001) (Figs. 4K-4O). The degree of brain injury in control animals was notable by gross examination of the brain tissue (Fig. 4P). Histological sections showed injury in the control group was widespread in all areas, where injury in the treated animals was significantly attenuated with limited focal injury in cerebral cortex and basal ganglia (Figs. 4P, 4Q). The hypoxic-ischemic injury score in the control group was significantly higher than in the treated group (overall injury score 27.7+ / -0.6 vs 11.0+ / -4.4, P<0.001, Fig. 4Q). Further, IVO2-treated swine had significantly less renal injury based on blood urea nitrogen (BUN) (Fig. 4R), creatinine (Fig. 4S), and histologic analysis. There were no significant differences in the lab or histologic manifestations of injury in other organs. These results demonstrate that oPMBs effectively reverse hypoxemia and, through a moderate increase in blood oxygenation, significantly decrease the burden of cardiac arrest, improve mortality, and diminish hypoxic-ischemic injury in acute, severe hypoxemia.

[0127] Fig. 4 shows that IVO2 via EmD oPMBs improved survival and meaningful outcomes in a swine model of severe hypoxemic-respiratory failure. Fig. 4A shows the study timeline. IVO2 treatment (oPMBs) (n=8) or control (DIO) solution (n=10) was administered at minutes 6, 8, and 10. Fig. 4B shows that IVO2 rapidly and significantly increased arterial oxyhemoglobin saturation (SaO2) during asphyxial period (gray shading). Fig. 4C shows that arterial carbon dioxide tension (pCO2) during asphyxia was significantly higher in IVO2- treated swine. Figs. 4B-4C show groups compared by two-way ANOVA, Sidak’s multiple

[0128] #14356316vl comparison with only significant P values shown. Fig. 4D shows that IVO2 treatment increased the fraction of animals free of cardiac arrest and cardiopulmonary resuscitation (CPR) during and post asphyxia period. Treatment period = shading. IVO2 treatment significantly decreased CPR time (Fig. 4E) and the required dose of epinephrine (FIG. 4F) used during resuscitation. Groups compared by student’s t-test. Fig. 4G shows that IVO2 treatment improved mean arterial blood pressure (MABP) during resuscitation. Group compared by two-way ANOVA, Sidak’s multiple comparison with only significant p values shown. Fig. 4H shows that IVO2 treatment significantly improved ROSC at 30 minutes (Log rank test P=0.003; Gehan-Breslow-Wilcoxon test, P=0.003) and 84-hour survival (Log rank test P=0.013; Gehan-Breslow-Wilcoxon test, P=0.007). Fig. 41 shows that IVO2 treatment significantly improved Swine Neurological Deficit Score (SNDS) in surviving swine. Groups compared by two-way ANOVA, Sidak’s multiple comparison. Fig. 4G shows that GFAP, a marker of astrocyte injury, was significantly elevated at day 3 in control group, whereas no difference from baseline was observed in treatment group. Groups compared by two-way ANOVA, Sidak’s multiple comparison test. Fig. 4K shows that representative weighted T2 MR image at 84 hours post-asphyxia depicted total grey matter and white matter diffusion restriction (supratentorial / infratentorial) with T2 prolongation throughout the cortex. Fig. 41 is a representative image in an IVO2-treated swine, revealing faint T2 prolongation in the basal ganglia. Figs. 4M and 4N show a three-dimensional representation of the median injury from brain MRI in control (Fig. 4M) vs treated (Fig. 4N) animals. Areas of enhancement on axial and coronal T2 and diffusion coefficient images were manually processed on a voxel- per- voxel basis. Fig. 40 shows that the volume of abnormal enhancement T2 and diffusion coefficient images was significantly lower in IV02-treated swine than in surviving control swine. Comparison by Student’s t-test. Fig. 4P shows that representative gross photos from the control group showed swollen, friable brain tissue with severe maceration of the ventral surface, and their pathological sections showed an overall dusky color, blurring of the gray white junction and intraventricular discoloration; in contrast, representative photos from the IV02-treated group revealed well-preserve brain tissue with few apparent abnormalities. Fig. 4Q shows that the histologic injury score was statistically significantly lower in the basal ganglia structures in IV02-treated swine than in controls. A score of 0 is no damage, 1: rare hyper eosinophilic neurons, 2: clusters of hyper eosinophilic neurons, 3: >50% of neurons are hyper eosinophilic, 4: >90% of neurons are hyper eosinophilic, 5: cavitated infarction. Groups compared by two-way ANOVA, Sidak’s multiple comparison test. Blood urea nitrogen (BUN) (Fig. 4R) and creatinine (Fig. 4S) were significantly higher in control group

[0129] #14356316vl on day 3 than in IVCh-treated swine. Note that Figs. 4I-4S reflects data collected only in surviving swine, which omits 7 of the 10 swine in the control group that did not survive. For all figures, data are means + / - SD, measurements are biological replicates.

[0130] EXAMPLE 5

[0131] Safety study. The treatment of swine with oPMBs was well tolerated and the animals showed no clinical sign of adverse effects. To probe the biodistribution and pharmacokinetics of the PMB constituents, positron emission tomography was performed with computed tomography (PET / CT) imaging for 7 days following injection of89Zr labeled LmD polymers in healthy rodents. Following a single tail vein injection (400 mg / kg, equivalent dose in swine study), the vast majority of LmD polymer was excreted in the urine within 24 hours, and the remainder underwent hepatic clearance (Figs. 5A-5D). By day 7, low levels of LmD were found in the spleen (5.9+ / - 1.5% injected dose per gram), liver (2.7+ / -0.7%) and kidneys (1.9+ / -0.5%). Based on empirical calculations

[0042] , >75% of injected polymer had been cleared, with the majority of the remainder visualized in the bowel lumen (i.e., in the process of being excreted) (Big. 5E). These results support the design hypothesis that the use of low MW and more hydrophilic polymers greatly facilitated clearance. To further assess the potential adverse effect of PMBs, a 14-day safety study was conducted in healthy rodents following a tail vein injection of a single dose of oPMBs (32 ml / kg, 70% foam, equivalent to the efficacy dose used above, n=12 oPMB, n=9 DIO control) with 4, 7, 14 day three time points, and a double dose administered 30 minutes apart (a total of 64 ml / kg, n=3); control animals (n=3) received equal volumes of D10. Throughout the observation period, all animals survived and exhibited normal behavior, had normal urine output, and weight gain (Eig. 5F). There were no differences between groups at any timepoint in blood gas, chemistry, complete blood count, or hepatic function testing (Figs. 5G-5O); there were no signs of clinically observable immune toxicities or platelet dysfunction. Further, rotational thromboelastometry (ROTEM) showed that PMBs did not affect either intrinsic or extrinsic coagulation pathways, with normal coagulation time, clot formation time, maximum clot firmness and maximum lysis at all time points (Figs. 5P-5W). Histological analysis of major organs in both groups showed similar and normal morphologies except that spleen macrophage vacuole formation was observed in treated animals; this was likely due to splenic polymer uptake, which interestingly was not observed in swine. These results together support that PMBs revert to soluble low MW molecular components after IV administration and are well-tolerated in clinically relevant doses.

[0132] #14356316vl Fig. 5 shows a biodistribution and safety study of LmD PMBs in rodents. Figs. 5A-5D show representative PET / CT images of rats receiving89Zr labelled polymers over time. Immediate after infusion, radioactivity in the upper abdomen, liver and kidneys, and a significant portion being excreted via bladder (Fig. 5A). Continuous excretion via urine and hepatic clearance at 24 hours (Fig. 5B). Bowel excretion continues via hepatic clearance at 48 hours, maximum accumulation in stools (Fig. 5C). Low radioactivity level on day 7 with continuous excretion via feces (Fig. 5D). Fige 5E shows the biodistribution on day 7, the residual polymer was presented as injected activity per gram of a particular organ (n=3, measurements are biological replicates). Figs. 5F-5O show major clinical markers for organ injury and toxicity were normal in animals receiving LmD PMBs compared with control group (n=3-6 per group) in rodent safety study. Dosage 1, 32 ml of 70% oPMBs (40 vol / vol% oxygen) per kg, equivalent to the efficacy dose, with endpoints at three time points (4, 7, and 14 days), whereas control groups receiving equal volume of DIO. Dosage x2, doubling of the dosage 1, administered 30 minutes apart, a total of 64 ml of 70% oPMBs (40 vol / vol% oxygen) per kg, 14-day single time point, whereas control group receiving equal volume of D10. Data are mean + / - SD, compared by multiple Mann-Whitney test. Liver function tests (Fig. 5G) Alkaline Phosphatase, (Fig. 5H) Alanine transaminase, (Fig. 51) Amylase. Renal function: (Fig. 5 J) BUN, (Fig. 5K) Creatinine. Lactate (Fig. 5L). Complete blood count: (Fig. 5M) Hemoglobins, (Fig. 5N) white blood cells, (Fig. 50) Platelets. All values of animals receiving PMBs of both dosages at all time points, were within normal ranges and showed no significant difference from control groups. Figs. 5P-5W show coagulation analysis by ROTEM in EXTEM and INTEM showed infusion of PMBs did not adversely affect clotting: (Figs. 5P, 5T) Clotting time, (Figs. 5Q, 5U) Clot formation time, (Figs. 5R, 5V) Maximum clot firmness, (Figs. 5S, 5W) Maximum lysis. Data are mean + / - SD, compared by multiple Mann- Whitney test with q value shown. All measurements are biological replicates.

[0133] EXAMPLE 6

[0134] Discussion. The above examples have described a pharmaceutically viable design of gas carrier to enable the clinical translation of IVO2 therapy. PMBs exhibit high gas carrying capacity, acceptable shelf stability, and manufacturability at sufficient scale and control to ultimately enable a clinical trial. At the gas concentration of the foams described (35-50 mL oxygen / dL foam), the provision of 100 mL oxygen gas required the co-administration of 100- 185 mL of additional fluid. Unlike prior injectable gas carriers (e.g., LOMs), the pH- triggered mechanism of PMBs actively enhanced their dissolution and the delivery of the gas

[0135] #14356316vl payload even in the absence of a diffusion gradient, hence minimizing the risk of vascular obstruction even when administered at a high dose or under conditions of low blood flow. This feature may be important to their use in settings of critical illness and emergencies in which cardiac output and blood flow may vary moment to moment, settings in which other gas carriers will cause gas embolism or particle jamming, pulmonary vascular obstruction, and cardiovascular collapse. Following dissolution, PMB shells rapidly reverted to low MW, soluble components, a strategy that decreases adverse non-specific hydrophobic interactions, a major contributor of nanotoxicity. These LmD PMBs showed high tolerance and undergo renal and hepatic clearance, a substantial improvement in safety from earlier generations. The polymeric structures of the shell materials may also be tunable. These may allow for the clinical intravenous injection of oxygen.

[0136] Acute, severe hypoxemia is a common and life-threatening event amongst critically ill patients and represents an enormous clinical challenge. Among others, it may occur in the setting of tracheal intubation, mechanical ventilation (e.g., due to secretions), or airway bleeding, and can lead to cardiac arrest when not immediately addressed. When hypoxia progresses beyond a critical threshold, mitochondrial reduction occurs, cells become energy deprived and morphologically damaged, and cardiovascular collapse ensues, and outcomes in such patients are dismal. It was shown that IVO2 may interrupt this lethal cascade for short periods of time until normoxia can be restored by conventional means. Preventing cardiovascular collapse in this setting is paramount for the prevention of neurologic injury. The brain is extremely oxygen avid and exquisitely sensitive to interruptions in oxygen supply, becoming isoelectric after 15-30 seconds absent blood flow and sustaining irreversible injury within minutes. In these examples, the initial, expected response to very severe hypoxia was a pronounced increase in blood pressure and heart rate (representing a sympathetic nervous system response), which degraded into hypotension and pulseless electrical activity as oxygen substrate became depleted. Intravenous oxygen is synergistically beneficial to the circulation in this setting: (1) local hyperoxia in the pulmonary arteries causes pulmonary vasodilation, lowering impedance to blood flow; (2) provision of oxygen for energy generation restores systemic vascular resistance (both arteriolar and venous) and myocardial function, raising perfusion pressure and blood flow; and (3) the residua of the polymer shell expand intravascular volume and myocardial preload, augmenting cardiac output. Together, these effects maintain or quickly restore the circulation in the setting of hypoxia-related cardiac arrest. Further, it was shown that the dose of oxygen required to accomplish these effects was a small fraction of baseline consumption in health. In illness

[0137] #14356316vl states such as severe hypoxia, oxygen consumption becomes supply-limited, i.e., such that the provision of a given dose of oxygen may have a more prolonged or pronounced effect on cellular metabolism. Further, a given dose of oxygen may also have a more pronounced effect when used in clinical settings of lung injury. Normally, oxygen flows from the alveolus into the blood, but in this model of airway obstruction, oxygen tension of the pulmonary artery exceeded that of the alveolus, such that oxygen initially diffused backwards, equilibrating with the functional residual capacity of the lung (i.e., increasing the volume of distribution of the gas pay load); in this sense, this was an exaggerated model of ventilation-perfusion inequality, a central pathology in patients with clinical lung disease. It is expected that this phenomenon of back-diffusion may be attenuated, and therefore the dose response may be more pronounced, in patients with more heterogenous ventilation-perfusion inequality or with an oxygen diffusion gradient. Taken together, these findings highlight the pharmacological advantages of IVO2 as a promising new treatment for the rapid reversal of life-threatening hypoxemia in emergency settings, including prehospital, intensive care unit, and operating room environments.

[0138] In clinical practice, it is envisioned that IVO2 would be available on-demand in environments caring for critically ill patients at risk for hypoxemia. Because arterial oxygen saturation is often continuously monitored by photoplethysmography in patients at risk for hypoxemia, IVO2 could become a new treatment for refractory hypoxemia that is refractory to current standards of care, including airway clearance (e.g., suctioning), lung recruitment (e.g., hand ventilation) and the use of other critical maneuvers. Its dosing could be titrated in the same way that pre-arrest bolus doses of epinephrine are titrated to treat refractory hypotension, since both blood pressure and arterial oxygen saturation (by plethysmography) are routinely measured as vital signs in hospitalized patients. As in the swine model, prespecified doses of IVO2 could be added to the resuscitation algorithm for patients being treated for in-hospital cardiac arrest (IHCA) caused by known or presumed hypoxemia; because such patients standardly receive IV resuscitative treatments within 3-5 minutes, IVO2 may restore early spontaneous circulation and significantly improve outcomes in such patients.

[0139] EXAMPLE 7

[0140] Below are materials and methods used in some of the above examples.

[0141] Polymer Synthesis. Dextran (MW 6 kD, 20 g) and 4-dimethylaminopyridine (64 g) were added to a round bottom flask under nitrogen and then dissolved in 200 ml of anhydrous DMSO via oil bath at 55 °C. Separately, 5.6 g of succinic anhydride and 20 ml of acetic

[0142] #14356316vl anhydride were dissolved in 60 ml of anhydrous DMSO and transferred to an additional funnel connected to the reaction flask. The anhydride solution was dropwise added to the reaction mixture over a period of 40 minutes under rigorous mixing using magnetic stirring. The reaction was maintained at 55 °C under nitrogen for 12 hours prior to work up. The reaction mixture was slowly precipitated in a 4 L beaker that contains 3 L 6% acetic acid aqueous solution under rigorous stirring. The resultant precipitate was collected by centrifuging and subsequently washed by ionized water three times. The final product was collected by freeze-drying as white powders.

[0143] Manufacturing of oxy gen-filled o-PMBs. All fabrication procedures were conducted inside biosafety workstation ISO class 5 equipped with vertical laminar flow (AirClean System). All equipment was UV sterilized and solutions were pre-sterilized by autoclave. The stock solution for homogenization was prepared as 1 Img / ml in 30 wt% dextrose solution via addition of 0.6 ml of IN sodium hydroxide per gram of polymer. Afterward, the LmD solution was placed on ice bath and a high-power UV lamp was submerged to sterilize the solution for 2 hours under magnetic stirring prior to homogenization. 90 ml of LmD stock solution was transferred into a 1 L beaker via a graduate cylinder, and the homogenization (L5M-A Laboratory Mixer, Silverson, USA) probe was submerged into the solution to stay at air-water interface. The beaker was placed in a water bath at 33 °C and the solution was let to equilibrated to the same temperature for 5 minutes. Afterward, the LmD solution was homogenized at 5.5 k RPM, upon homogenization the solution immediately turned into a viscous white foam. The beaker position was occasionally adjusted manually to maintain maximum and even mixing. The polymer solution was first mixed for 2 minutes, followed by addition of 0.24 ml of dilute hydrochloric acid (HC1) (0.6%) by pipetting, the homogenization process continues with addition of the same amount of HC1 every minute for another 8 minutes (a total of 9 additions of acids). The homogenization continued for another minute and terminated at 11th minute. Upon the last addition of acid (~10 min), a notable decrease in foam viscosity can be observed, suggesting the solution pHs transitioned below pKa to protonate majority of carboxylic acid groups, and the polymer cross-linking led to a phase transition. The resulting foam was left undisturbed on the water bath for another 10 minutes, afterwards 150 ml of 10% dextrose solution was poured onto the foam to help transfer the foam mixture from the beaker to a 500 ml conical shaped flask. Multiple batches of LmD foams were combined and collected into conical-shaped flasks. They were allowed to sit overnight, and LmD PMBs floated to the top to form a cake-like cream layer, while polymeric debris accumulated in the bottom flask. Then the bottom fluid of foam as well as

[0144] #14356316vl polymer debris were siphoned using a roller pump and a long stainless- steel needle, and then fresh sterile DIO solution was added to the thick foam layer and re-disperse them in solution via gentle shaking of the flask. This process was repeated 3 times, and foam layers from various batches were further combined and concentrated to the desired final concentrations. To oxygenate the PMBs, the PMB foams were placed in a flask with a silicon septum, and the headspace was then purged by flowing humidified oxygen via a 0.25-micron sterile filter for 12 hours with occasional shaking of the flask. A small aliquot of the solution was drawn to a syringe via a sterile needle to measure the pCL of the solution. The fully oxygenated PMBs were then transferred and constituted as desirable concentration in 60 ml syringes prior to intravenous administration for animal experiments. Sterility tests were conducted throughout the entire fabrication process. Both before and after the PMB fabrication, the sterility of each LmD solution, each fabrication batch as well as combined foams were tracked and tested by plating onto blood agar plates which were continuously monitored for potential bacterial growth. Only lots that exhibited no colony growth after incubation for 7 days were used for animal experiments.

[0145] All animal experiments conducted in this work were approved and conducted according to Boston Children’s Hospital Institutional Animal Care and Use Committee (IACUC) policy.

[0146] Rodent hemodynamic safety studies. Male Sprague-Dawley rats (weight 503+ / -52 g, Charles River Laboratories) were induced for anesthesia with intraperitoneal injection of ketamine (45-75 mg / kg) and xylazine (5-10 mg / kg) followed by orotracheal intubation. Then, anesthesia was maintained by inhalational isoflurane (1-2%). Animals were mechanically ventilated (SAR-1000, CWE Inc, Birmingham, Alabama) with a tidal volume 5-8 mL / kg, respiratory rate 40-45 breaths / minute on 30% oxygen. Instrumentation included 24 G angiocatheter in the tail vein for microparticle infusion, two femoral artery cannulae placed by cutdown, one with a 24 G angiocatheter used for blood collection and the other with a pressure catheter for hemodynamic monitoring (Millar Mikro-Tip Pressure Catheter Transducer, model SPR-671, 1.4F), femoral venous catheter (3 French) placed by cutdown and advanced to the right atrium to monitor central venous pressure. Following, a median sternotomy was performed, and pressure-volume catheters (Millar Mikro-Tip Pressure- Volume Catheter Transducer, 9 mm spacing, model SPR-847, 1.4F) were place into the right and the left ventricles, for pulmonary artery pressure and cardiac output monitoring, respectively. Temperature was controlled by a rectal temperature probe connected to a heating pad for a central temperature target of 37 °C. All these instruments were calibrated

[0147] #14356316vl and continuously recorded using PowerLab / LabChart software (LabChart Pro 8 software, ADInstruments). After a 15-minute baseline period, animals were injected with either intravenous oxygen formulation (IVO2) (80% foam) or control solution (10% dextrose). Each injection was a total of 5 mL volume over 1 minute, followed by a 1 mL Plasma-Lyte flush, and then a 2-minute stabilization period. Each injection was repeated a total of 5 times. Afterwards, there was a 60-minute observation period with continuous hemodynamic monitoring. Animals were euthanized at the end of the study by cardiac explantation. Mean arterial blood pressure (MABP), left ventricular end-diastolic pressure (LVEDP), cardiac index (CI= stroke volume*heart rate / kg), and pulmonary vascular resistance (PVR = [mean pulmonary arterial pressure-LVEDP] / CI) were exported as 1-minute averages. Baseline was calculated as the average of the 15-minute baseline in all animals included in the study, then data is presented as percentage change from baseline. Comparison between groups was performed analyzing averages over the 3 periods by ANOVA.

[0148] Rodent echocardiography safety study. Male Sprague-Dawley rats (weight 400-500 g, Charles River Laboratories) were instrumented similarly to our previous study, including orotracheal intubation, mechanical ventilation (FiO20.3), femoral vein catheterization and femoral artery hemodynamic continuous monitoring. Anesthesia was maintained with inhaled isoflurane (1-2%). Transthoracic echocardiography (Philips EPIQ ultrasound machines, Philips Healthcare, Andover, Massachusetts) was performed by a certified Pediatric Cardiologist in the left parasternal window for a four-chamber view. Our intravenous formulation filled with either oxygen (IVO2) or air gas (IV Air) and a previous generation of lipid oxygen microparticles (LOM) were compared to a control echocardiography where no infusion was performed. Injections at three different rates (4 mL / kg / min, 8 mL / kg / min and 12 mL / kg / min) were analyzed by ImageJ (US National Institutes of Health, Maryland) for intensity quantification. A selection of 10 frames per study were randomly selected and compared to control images. Left and right ventricle cavity areas were selected in the analyzer and intensity quantified relative to the ventricle surface area. Results were compared by ANOVA.

[0149] Efficacy study in asphyxia model in swine. Female Yorkshire swine (10.1-12.5 kg, Parson’s Farms) were housed individually within a 12-hour dark-light cycle with free access to food and water. At least a week of acclimatization was maintained before the experiment.

[0150] Experimental protocol. Animals were anesthetized by intramuscular injection of tiletamine and xylazine and orally intubated via direct laryngoscopy. Following endotracheal intubation, the animal was connected to a mechanical ventilator and sedated with isoflurane

[0151] #14356316vl 1-3%, titrated to effect. Ventilation was managed on volume control, with tidal volumes 8-10 mL / kg, PEEP 5, rate 12-15 bpm, and FiO221% (Draeger Apollo). Minute ventilation was titrated to achieve an end tidal carbon dioxide concentration of 40 mmHg. Core temperature was maintained at 38 °C with a heating blanket. Oxygen consumption was monitored during the baseline period (GE E-CAIOVx Respiratory Module). Instrumentation included a femoral arterial catheter for blood sampling (3 French, 5 cm) and a femoral venous catheter for treatment / control infusion (3 French, 5 cm). An oximetric catheter (PediaSat, 4.5 Fr, 5 cm, Edwards Eifesciences Corporation, California) was also placed in the contralateral femoral artery (for continuous hemodynamic monitoring) and in the right internal jugular vein. All catheters were placed by surgical cutdown using Seidinger technique under direct visualization of the vessels. Catheters were transduced and calibrated according to manufacturer’s instructions prior to use. Swine were monitored continuously with telemetry, pulse oximetry, and cerebral and somatic near-infrared reflectance spectrometry (NIRS, Somanetics). CPR quality was monitored using real-time feedback (CPR Electrodes, M Series, Zoll Medical Corporation) for real-time feedback and defibrillation. Following instrumentation, anesthesia was transitioned to intravenous fentanyl (20 mcg / kg / dose) and cisatracurium (0.5 mg / kg / dose) and isoflurane was discontinued for the remainder of the experiment. Inhalational anesthesia was discontinued 30 minutes prior to asphyxia.

[0152] Anesthetic level was monitored by hemodynamic response and movement to painful stimuli, and bolus doses repeated as needed. Following a 30 minute baseline period, baseline blood samples were obtained for complete blood count (CBC), biochemistry, venous and arterial blood gases, and serum for neuro biomarkers.

[0153] Following baseline observation period, the endotracheal tube was clamp-occluded, and the ventilator disconnected (Time=0). Arterial blood gases were obtained every 2 minutes (starting on Time=l) during the first 30 minutes. Cardiac arrest was defined as a systolic blood pressure (SBP) less than 40 mmHg for 5 seconds or longer. When in cardiac arrest, animals received metronome-guided, high-quality chest compressions, and intravenous medications as outlined by the American Heart Association Advanced Cardiac Fife Support (ACES) algorithm, including Epinephrine 0.01 mg / kg every 2 minutes, Eidocaine 1 mg / kg, Atropine 0.01 mg / kg, Amiodarone 5 mg / kg, calcium gluconate 50 mg / kg, and sodium bicarbonate (as needed, 1 mEq / kg). Animals were randomly allocated to an intervention, either intravenous oxygen or control. Intravenous oxygen consisted of 400 ml of 60% PMBs (-140 ml oxygen and 260 ml D10). Control consisted of 260 ml D10 solution, at 13 mL / kg

[0154] #14356316vl per dose. At minutes 6, 8 and 10, the allocated intervention was administered via the femoral CVC. Each of the three doses were administered over 2 minutes.

[0155] At time=12 minutes, the tracheal tube clamp was removed, and ventilation was restored with 100% oxygen and the ventilator settings above (Maquet Servo i). CPR was continued until Time=30 minutes or until return of spontaneous circulation (ROSC), defined as SBP>50 mmHg.

[0156] Survival period and critical care metrics. Surviving animals were then maintained in an intensive care environment for 4 days. Animals were mechanically ventilated and assessed for extubation readiness every 12 hours with a spontaneous breathing test as well as neurological and hemodynamic status. Inotropic support with continuous infusions of dopamine (3 to 10 mcg / kg / min), epinephrine (0.02 to 0.2 mcg / kg / min), norepinephrine (0.02 to 0.5 mcg / kg / min) and vasopressin (0.0005 to 0.002 U / kg / min) were used as needed to maintain SBP >70 mmHg. Sedation and analgesia were maintained while intubated with continuous infusions of propofol (1 to 3 mg / kg / h) and fentanyl (5 to 10 mcg / kg / h) as needed for animal comfort. Complete neurologic examination using a previously validated tool, Swine Neurological Deficit Score (SNDS) was performed at baseline, T=6 hours, and daily thereafter. This score includes an evaluation of cranial nerves, respiration, motor and sensory function, level of consciousness and behavior. In this score, 0 is considered normal (no deficits) and 500 is equivalent to brain death.

[0157] A determination of extubation readiness was made on the following conditions: no seizures in the past 2 hours, not on inotropic support, respiratory rate <40 breaths / minute and SpO2 >92% on pressure support only ventilation (PS 6, PEEP 5, FiO2<50%) for 2 hours. If the animal was deemed to be clinically ready to extubate, all lines were removed except for the internal jugular catheter, which was maintained for blood sampling and drug administration. A fentanyl patch (25 mcg) was placed for analgesia. Once extubated, the animal was transferred to a kennel for observation and monitoring with continuous pulse oximetry.

[0158] Seizures were treated as follows. Airway and hemodynamic support were provided as clinically indicated. If a seizure lasted for >2 minutes, a dose of IV lorazepam was administered (0.1 mg / kg) every 5 minutes up to 3 doses. Thereafter, persistent seizures were treated with IV phenytoin (20 mg PE / kg), repeated once after 15 minutes if seizures persisted. Thereafter, persistent seizures were treated with IV phenobarbital 20 mg / kg, repeated once after 15 minutes if seizures persisted. Swine who were persistently seizing thereafter were treated with an infusion of midazolam, with a starting dose of 0.1 mg / kg / hour, with an hourly

[0159] #14356316vl uptitration of 0.1 mg / kg / hour to a maximum dose of 0.7 mg / kg / hour. Inotropic support was provided as required for hypotension.

[0160] Diabetes insipidus was treated as follows. If urine output exceeded 3 mL / kg / hour, serum sodium was checked every 2 hours. When sodium exceeded 150 mmol / L and UOP exceeded 5 mL / kg / hour for 2 hours, vasopressin infusion was initiated at 1 milliunit / kg / hr and uptitrated hourly to 10 milliunit / kg / hr until UOP decreased to <5 mL / kg / hour. During the survival period, the following monitoring labs were performed at 30 minutes, 1, 2, 3, 4, 6, 12, 24 hours and then twice daily during the survival period: blood gas, CBC, biochemistry, glial fibrillary acidic protein (GFAP) in serum. GFAP was assessed using an electrochemiluminescent immunoassay (Meso Scale Diagnostics, Rockville, Maryland), with a detection range of 0.001 to 40.0 ng / ml.

[0161] Brain magnetic resonance imaging. On day 4, animals were reintubated if they had been previously extubated and anesthetized for brain magnetic resonance imaging (MRI) (3 Tesla Skyra model scanner, 64-channel head and neck coil, Siemens Corporation, Germany). High resolution images including weighted Tl, T2, magnetic resonance angiography (MRA), diffusion weighted imaging (DWI), susceptibility weighted imaging (SWI), magnetic resonance spectroscopy (MSR), and fluid attenuated inversion recovery (FLAIR) were obtained. These images were analyzed by a board-certified neuroradiologist blinded to treatment allocation. Then, areas of enhancement on axial and coronal T2 and diffusion coefficient images were manually processed on a voxel-per- voxel basis and outlined (itk-SNP software application, Penn Image Computing and Science Laboratory, University of Pennsylvania, Pennsylvania, and Scientific Computing and Imaging Institute, University of Utah, Utah) by the Department of Radiology at Boston Children’s Hospital, all of whom were also blinded to treatment allocation. From these values, total volumes of cranial injury were calculated using software normalized to brain volume. The total volume of injury was compared between groups by t-test.

[0162] Neurohistology analysis. Following brain MRI, both internal jugular veins and carotids were cannulated by cutdown for brain perfusion. The brain was perfused by administrating 2 L of normal saline via both carotid arteries followed by 4 L of paraformaldehyde for fixation. During this procedure the animal was sedated with inhaled isoflurane until death was confirmed by absence of vital signs. A complete autopsy was performed including all vital organs and brain. All organs were formalin-fixed prior to pathological analysis. The skull of swine was opened, and the head was fixed in 4% paraformaldehyde for 72 hours, following which brain tissue was extracted and placed in

[0163] #14356316vl formalin for analysis. Brains were processed, sliced, embedded in paraffin, and stained for hematoxylin and eosin, and slides were examined by fluorescence with a Rhodamine filter. Hypoxic-ischemic injuries were studied in 7 different regions: frontal, parietal and temporal cortex, dentate gyrus and pyramidal layer of hippocampus, caudate and thalamus, by a board- certified pathologist, who was blinded to treatment allocation. The lesions were graded according to a previously defined scale as follows: 0 = no injury, 1 = rare hypereosinophilic (HE) neurons, 2 = clusters of HE neurons, 3 = over 50% of HE neurons, 4 = over 90% HE neurons, 5 = cavitated infarction. This analysis only included animals that survived during the 4-day observation period. Heart, lungs, kidneys, and liver were subjectively reviewed by a pathologist blinded to treatment group.

[0164] PET / CT imaging and Biodistribution study. 400 mg of LmD polymer was dissolved in 10 ml water by adjusting pH to 6 in a round bottom flask, to which TCO-PEG3-Amine (8 mg, 0.021mmol, Click Chemistry Tool, USA), l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (5.76 mg, 0.03 mmol) and N-hydroxy succinimide (2.3 mg, 0.02 mmol) were added to react at room temperature. After 12 hours, the product was precipitated by adding dilute hydrochloride acid (0.1M) and centrifuged and repeatedly washed with water. The final product LmD-TCO was collected as solid powder. Separately, tetrazine-NHS ester (0.12 mmol, 38 mg) and DFO-NH2 / mesylate (69 mg, 95%, 0.1 mmol) were reacted in 1 ml of DMF in the presence of 30 pl of triethylamine for 12 hours at room temperature, after which, the product tetrazine-DFO was precipitated with excess of acetone and obtained as a pink powder after drying. Chemicals and reagents were obtained from Sigma- Aldrich (St Louis, MO) unless otherwise specified. To minimize metal contamination, glassware was washed with 2 M nitric acid (Fisher Scientific, Fair Lawn, NJ: certified ACS plus grade) and rinsed with ultrapure water (>15 MQ resistivity) (US Filter / Siemens Water Technologies, Warrendale, PA) before use. All solutions were prepared using ultrapure water. Metal contaminants were removed from buffer solutions using a Chelex-100 resin column (Bio-Rad Laboratories, Hercules, CA). Metal-naive pipette tips were purchased from Rainin Instrument (Oakland, CA) and were used to prepare all samples. Plasticware was washed with ultra-pure water and then equilibrated with buffers where appropriate. Zirconium-89 was purchased from Washington University in St. Louis (St. Louis, MO) and was supplied in 1 M oxalic acid. In a representative labeling reaction, a 40 microliter aliquot of the stock89Zr solution (in 1 M oxalic acid) was neutralized with 18 microliter 2 M Na2CO3, 100 microliter PBS was added, and the pH was adjusted to 7 with glacial acetic acid. Then 0.1 ml of 1 mg / ml of tetrazine-DFO in DMSO was added into the tube and the solution was incubated at

[0165] #14356316vl room temperature for 1 hour. Afterwards 1 ml of LmD-TCO (20 mg / ml) in PBS solution (pH adjusted to 7.2) was added and allowed to react with the89Zr-complexed tetrazine-DFO for 5 minutes. The reaction was monitored by ITLC, whereas89Zr-LmD complexes remained at the origin and unbound89Zr migrates with the solvent front. To remove unbound89Zr, the labeled LmD polymers were precipitated by adding 0.1 ml of acetic acid, and the labeled product was collected by centrifugation. The labeled polymer was further purified by repeatedly washing with water and centrifuging. The final polymers were then redissolved in PBS buffer by adding IN sodium hydroxide to adjust the pH to 7.2 at a concentration between 5 to 10 mg / ml.

[0166] Small-animal PET / CT image data were obtained using a Bruker Albira multimodality (PET / SPECT / CT) small-animal imaging system (Bruker Corporation; Woodbridge CT). Phosphor storage screens were scanned using a Fujifilm BAS-5000 (FUJIFILM Life Science, Stamford CT). Radioactivity in the tissue samples was assayed with a Packard Cobra II automated gamma counter (Meriden, CT). Once the polymer was radiolabeled, the injection solution was prepared by mixing between 5-10 mg labeled polymer (radioactivity of between 120 to 140 microcurie) with unlabeled (cold) polymers in a total of 6~7 ml of PBS solution. Male Sprague Dawley rats (125-150 g) were anesthetized on 2% isoflurane via nose cone, and the prepared polymer solution administered via tail- vein injection (400 mg / kg). Afterwards, PET / CT imaging was performed immediately after injection, 3, 24, 48 hours and then every 48 hours until day 7. On day 7 after injection, animals were euthanized by cardiac explantation under anesthesia (3% isoflurane). Tissue samples were immediately collected and weighed and then assayed by gamma counter to determine89Zr activity. The percent of injected dose per tissue gram was calculated by comparison to samples of known89Zr concentration.

[0167] Rodent 14-day safety study. Male Sprague-Dawley rats (400-500 g) were induced for anesthesia with 1 to 2% isoflurane, inhaled via nose cone. A tail vein catheter was placed, flushed with Plasma- Lyte and then, animals were randomized to either receive intravenous oxygen solution (treatment, 70% foam) or 10% dextrose solution (control) at a single dose 32 mL / kg, over 10 minutes, followed by a 1 mL Plasma-Lyte flush, or a double dose (a total of 64 ml / kg, 30 minutes between each dose), n=3-6. After recovering from anesthesia, animals were placed in metabolic cages for 6 hours for urine and stool output recording. In metabolic cages, animals had access to food but not to water. This process was repeated once daily. Animals’ behavior was monitored daily, including activity, response to pain, response to light and sound, respiration pattern and comfort. Randomly at 3, 7 or 14 days, animals were

[0168] #14356316vl anesthetized with isoflurane, a catheter was placed in the femoral artery for blood collection including blood gas analyzed by ABL 90 Flex Plus (Radiometer America, California), complete blood count (CBC) analyzed by VetScan (HM5 Hematology Analyzer, Zoetis, New Jersey), basic chemistry panel analyzed by VetScan (VS2 Chemistry Analyzer, Zoetis, New Jersey) and coagulation analyzed by Rotational Thromboelastometry (ROTEM Delta 3678, Massachusetts) and then euthanized by heart explantation, collecting vital organs. Heart, lungs, kidneys, and liver were placed in formalin and then sent to the Department of Research Histopathology at Harvard Medical School for blind analysis. Organs were processed, sliced, embedded in paraffin, and stained for hematoxylin and eosin.

[0169] Statistical analysis. The primary outcome of this study was Swine Neurologic Deficit Score (SNDS) at 3 days in surviving swine. Based on the preliminary data, a Swine Neurologic Deficit Score of 420 with SD of 100 in surviving swine in the control group was expected. The inclusion of 3 surviving animals per group allowed the detection of a difference of 250 in the SNDS at 3 days with 90% power and alpha of 0.05, which would be very clinically significant. Continuous variables were described as mean and SD, and categorical were described as number and percentages. Differences between groups in arterial saturation, arterial carbon dioxide tension, hemodynamics, histopathology scores, chemistry and hematologic parameters were evaluated over time by using a 2-way repeated measures analysis of variance (ANOVA) with an interaction term between treatment group and time; time-dependent differences between groups were evaluated using a post-hoc analysis (Sidak multiple comparison method). Any missing values were imputed as median values across the group for that time point. Single time point values, such as baseline characteristics, resuscitation outcomes, GFAP changes and differences in cerebral infarct volumes, were compared between groups by Student’s t-test, Mann- Whitney U test or exact Fisher test, as appropriate. Statistical analysis and graphing were performed by Prism version 9.00 software (GraphPad, California). For all test, a 2-sided p value <0.05 was considered statistically significant.

[0170] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations

[0171] #14356316vl described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0172] In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0173] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0174] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0175] #14356316vl As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0176] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0177] When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0178] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0179] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,”

[0180] #14356316vl “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0181] #14356316vl

Claims

CLAIMSWhat is claimed is:

1. A composition, comprising: particles having a shell surrounding a gas core, the particles having an average size of less than 10 micrometers, the shell comprising cross-linked dextran having a molecular weight of less than 40 kDa, wherein the dextran is succinylated and / or acetylated.

2. The composition of claim 1, wherein the particles have an average size of less than 5 micrometers.

3. The composition of any one of claims 1 or 2, wherein the dextran is water-soluble.

4. The composition of claim 3, wherein the water-soluble dextran, prior to cross-linking, exhibits a mean hydrodynamic radius less than 20 nm.

5. The composition of any one of claims 3 or 4, wherein the water-soluble dextran, prior to cross-linking, exhibits a mean hydrodynamic radius less than 15 nm.

6. The composition of any one of claims 1-5, wherein the dextran is amphiphilic.

7. The composition of any one of claims 1-6, wherein the dextran has an acetyl substitution of between 1 and 3.

8. The composition of any one of claims 1-7, wherein the dextran has a succinyl substitution of between 0 and 2.

9. The composition of any one of claims 1-8, wherein the dextran has a molecular weight of between 500 Da and 40 kDa.

10. The composition of any one of claims 1-9, wherein the dextran has a molecular weight of less than 12 kDa.#14356316vl11. The composition of any one of claims 1-10, wherein the dextran has a molecular weight of between 6 kDa and 10 kDa.

12. The composition of any one of claims 1-11, wherein the dextran is cross-linked via carboxylate groups.

13. The composition of any one of claims 1-12, wherein the gas comprises air.

14. The composition of any one of claims 1-13, wherein the gas comprises at least 30 vol% O2.

15. The composition of any one of claims 1-14, wherein the particle consists of a single shell surrounding a gas core.

16. The composition of any one of claims 1-15, wherein the shell does not comprise a particle aggregate.

17. The composition of any one of claims 1-16, wherein the dextran has a structure:wherein n is a positive integer less than 100.

18. A composition, comprising: particles having a shell surrounding a gas core, the particles having an average size of less than 10 micrometers, the shell comprising a water-soluble polymer that exhibits cross-linking at a pH of less than 6.

19. The composition of claim 18, wherein the polymer comprises dextran.#14356316vl20. The composition of claim 19, wherein the dextran is succinylated and / or acetylated.

21. The composition of any one of claims 18-20, wherein the polymer comprises starch.

22. The composition of claim 21, wherein the starch has a structure:wherein n is a positive integer less than 100.

23. The composition of any one of claims 18-22, wherein the particles have an average size of less than 5 micrometers.

24. The composition of any one of claims 18-23, wherein the gas comprises air.

25. The composition of any one of claims 18-24, wherein the gas comprises at least 30 vol% O2.

26. A method, comprising: creating bubbles of gas in a solution comprising a succinylated and / or acetylated dextran having a molecular weight of less than 40 kDa; and acidifying the solution to a pH of less than 7 to absorb the dextran onto the bubbles of gas and cause cross-linking of the dextran.

27. The method of claim 26, wherein the bubbles of gas have an average diameter of less than about 10 micrometers within the solution.

28. The method of any one of claims 26 or 27, wherein the bubbles of gas have an average diameter of less than about 5 micrometers within the solution.#14356316vl29. The method of any one of claims 26-28, comprising absorbing dextran on the bubbles such that the dextran forms a coating having a cross-sectional thickness of less than 50 nm on the bubbles.

30. The method of any one of claims 26-29, wherein the bubbles comprise air.

31. The method of any one of claims 26-30, wherein the gas comprises at least 30 vol% O2.

32. The method of any one of claims 26-31, wherein the dextran is water-soluble.

33. The method of claim 32, wherein the water-soluble dextran, prior to cross-linking, exhibits a mean hydrodynamic radius less than 20 nm.

34. The method of any one of claims 32 or 33, wherein the water-soluble dextran, prior to cross-linking, exhibits a mean hydrodynamic radius less than 10 nm.

35. The method of any one of claims 26-34, wherein the dextran is amphiphilic.

36. The method of any one of claims 26-35, wherein the dextran exhibits an acetyl substitution of between 1 and 3.

37. The method of any one of claims 26-36, wherein the dextran exhibits a succinyl substitution of between 0 and 2.

38. The method of any one of claims 26-37, wherein the dextran has a molecular weight of between 500 Da and 40 kDa.

39. The method of any one of claims 26-38, wherein the dextran has a molecular weight of less than 12 kDa.

40. The method of any one of claims 26-39, wherein the dextran has a molecular weight of between 6 kDa and 10 kDa.#14356316vl41. A method, comprising: creating bubbles of gas in a solution comprising a water-soluble polymer having a molecular weight of less than 60 kDa; and acidifying the solution to a pH of less than 7 to absorb the water-soluble polymer onto the bubbles of gas.

42. The method of claim 41, wherein the polymer is pH-responsive.

43. The method of any one of claims 41 or 42, wherein the bubbles of gas have an average diameter of less than about 10 micrometers within the solution.

44. The method of any one of claims 41-43, wherein the bubbles of gas have an average diameter of less than about 5 micrometers within the solution.

45. The method of any one of claims 41-44, comprising absorbing the polymer on the bubbles such that the polymer forms a coating having a cross-sectional thickness of less than 50 nm on the bubbles.

46. The method of any one of claims 41-45, wherein the bubbles comprise air.

47. The method of any one of claims 41-46, wherein the gas comprises at least 30 vol% O2.

48. The method of any one of claims 41-47, wherein the water-soluble polymer exhibits a mean hydrodynamic radius less than 20 nm prior to acidifying the solution.

49. The method of any one of claims 41-48, water-soluble polymer exhibits a mean hydrodynamic radius less than 20 nm prior to acidifying the solution.

50. The method of any one of claims 41-49, water-soluble polymer exhibits a mean hydrodynamic radius less than 15 nm prior to acidifying the solution.

51. The method of any one of claims 41-50, wherein the polymer is pH-responsive.#14356316vl52. The method of any one of claims 41-51, wherein the polymer comprises dextran.

53. The method of claim 52, wherein the dextran is succinylated and / or acetylated.

54. The method of any one of claims 41-53, wherein the polymer comprises starch.

55. The method of any one of claims 41-54, wherein the starch comprises an acetyl group.

56. The method of any one of claims 41-55, wherein the starch comprises an alkyl group.

57. The method of any one of claims 41-56, wherein the starch comprises a carboxylic acid group.

58. The method of any one of claims 41-57, wherein the polymer has a molecular weight of between 500 Da and 40 kDa.

59. The method of any one of claims 41-58, wherein the polymer has a molecular weight of less than 12 kDa.

60. The method of any one of claims 41-59, wherein the polymer has a molecular weight of between 6 kDa and 10 kDa.#14356316vl

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