Prodrug copolymers and polymeric micelles thereof for delivery of short chain fatty acids, promotion of gut health, and treatment of disease - Patents.com

Abstract

Provided herein are polymeric materials that find use in, for example, the delivery of short chain fatty acids. In particular, provided are polymers that form stable nanoscale structures and release their payloads, for example, by cleavage of covalent bonds (e.g., by hydrolysis or enzymatic cleavage). The polymers are useful, for example, for the delivery of payloads (e.g., short chain fatty acids (SCFAs)) to the intestine for health and disease treatment applications, and have broad applicability in diseases associated with alterations in the human microbiota, including inflammatory diseases, autoimmune diseases, allergic diseases, metabolic diseases, and central nervous system diseases, among others.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 329,913, filed April 12, 2022, and U.S. Provisional Patent Application No. 63 / 275,260, filed November 3, 2021, both of which are incorporated herein by reference.

[0002] Provided herein are polymeric materials that find use, for example, in the delivery of short-chain fatty acids. Copolymers are provided that form stable nanoscale structures (e.g., micelles) and release their payloads, for example, by cleavage of covalent bonds (e.g., by hydrolysis or enzymatic cleavage). The polymers are useful, for example, for delivering payloads (e.g., short-chain fatty acids (SCFAs)) to the intestine for health and disease treatment applications, with broad applicability to diseases associated with alterations in the human microbiota, including inflammatory diseases, autoimmune diseases, allergic diseases, metabolic diseases, and central nervous system diseases, among others. In certain embodiments, provided herein are prodrug polymeric micelles that find use in delivering short-chain fatty acids to the intestine for promoting gut health, establishing a healthy microbiota, and treating immune and / or inflammatory diseases such as inflammatory bowel disease and food allergies. [Background technology]

[0003] The gut microbiota has many effects on both mucosal and systemic health (Reference B9; incorporated by reference in its entirety). Resident bacteria play a key role in maintaining mucosal homeostasis, in part through the production of short-chain fatty acids, particularly butyrate esters (References B10-B12; incorporated by reference in their entirety). Butyrate esters are produced by a subset of gut bacteria through the fermentation of dietary fiber (Reference B13; incorporated by reference in its entirety). Butyrate esters are the preferred energy substrate for colonic epithelial cells and enhance intestinal barrier function by stabilizing hypoxia-inducible factors and maintaining epithelial tight junctions (References B12, B14; incorporated by reference in their entirety). Butyrate esters also promote the production of antimicrobial peptides (AMPs), which regulate gut homeostasis by shaping the composition of the microbiota (Reference B15; incorporated by reference in its entirety). To mediate its immunomodulatory functions, butyrate acts via signaling through specific G protein-coupled receptors or as an inhibitor of histone deacetylase activity (HDAC) (Reference B5; incorporated by reference in its entirety). HDAC inhibition by SCFAs promotes the differentiation of colonic regulatory T cells (Tregs) (References B16-B18; incorporated by reference in their entireties).

[0004] Food allergy is a common and severe disease affecting more than 32 million Americans (Reference B7; incorporated by reference in its entirety). Among adults in the United States with food allergies, 38% reported at least one food allergy-related emergency department visit in their lifetime (Reference B7; incorporated by reference in its entirety). Recently, Palforzia was approved by the U.S. FDA as oral immunotherapy (OIT) for peanut allergy, becoming the first approved treatment for food allergy (Reference B7; incorporated by reference in its entirety). The goal of this therapy is to establish a state of desensitization by exposing patients to gradually increasing doses of peanut protein. OIT has shown efficacy in inducing desensitization to peanut antigens, but requires chronic dose increases while gastrointestinal symptoms are common (Reference B7; incorporated by reference in its entirety). Furthermore, OIT, in its current form, is unlikely to achieve long-term, sustained nonresponsiveness to peanut antigens. 8 Due to the adverse effects and limited effectiveness of OIT, there is an urgent need to develop new therapies for food allergies.

[0005] Experiments have shown that neonatal administration of antibiotics reduces gut microbial diversity and impairs epithelial barrier function, resulting in increased access of food allergens to the systemic circulation (Reference B19; incorporated by reference in its entirety). Administration of a consortium of spore-forming bacteria from the class Clostridium restored epithelial barrier integrity and prevented allergic sensitization to food (Reference B19; incorporated by reference in its entirety). Experiments have further demonstrated the causal role of bacteria present in the microbiota of healthy infants in protection against cow's milk allergy (Reference B3; incorporated by reference in its entirety). Germ-free (GF) mice, which are protected from anaphylactic reactions to cow's milk allergens, are transferred with microbiota from healthy, but not cow's milk allergic (CMA) human infants. By integrating the differences between the changes in ileal gene expression induced by colonization of GF mice and the microbiota signature present in the healthy CMA microbiota, a single butyrate-producing Clostridium species, Anaerostipes caccae, was identified, which mimicked the effects of a healthy microbiota during monocolonization of GF mice (Reference B3; incorporated by reference in its entirety). Recent findings from diverse cohorts of pediatric and adult twins on food allergy, validating mouse model data with human microbiota samples, were both concordant and discordant. Most of the differentially abundant taxonomic operational units (OTUs) between healthy and allergic twins were found to reside in the Clostridia class, and a study of twins across a wide age range demonstrated that early-life depletion of allergy-protective Clostridia is maintained throughout life (Reference B2; incorporated by reference in its entirety). Although there is significant interest in the use of butyrate-producing clostridia as biotherapeutics, long-term engraftment of oxygen-sensitive anaerobic bacteria has proven difficult (see references B20-B21; incorporated by reference in their entirety).

[0006] Butyrate esters, produced by bacteria via metabolic dietary fiber, are known to be agonists of G protein-coupled receptors and inhibitors of histone deacetylases (HDACs) (Reference A1; incorporated by reference in its entirety). Butyrate esters are also preferred substrates for intestinal epithelial cells (Reference A2; incorporated by reference in its entirety), stabilizing hypoxia-inducible factors and maintaining tight junctions to enhance intestinal barrier function (Reference A3; incorporated by reference in its entirety). In addition, butyrate esters have been demonstrated to induce colonic regulatory T cells (References A4-A6; incorporated by reference in their entirety). The important role that butyrate esters play in intestinal immunity makes them good candidate drugs for protecting intestinal immunity and inducing oral tolerance. However, butyrate esters and other short-chain fatty acids are not suitable for oral administration. Even when enteric-coated or encapsulated, butyrate esters have an unpleasant and persistent odor and taste. Orally administered butyrate esters as the sodium salt, which may have therapeutic effects, are not absorbed in parts of the intestine and are metabolized too rapidly to maintain pharmacological effects. 22 Previous studies in mouse models demonstrating the therapeutic effects of butyrate esters were based on high-concentration, ad libitum exposure to butyrate esters (mM amounts in drinking water) or the use of butyrate-esterified starch (see References B16-B18, B23-B25; incorporated by reference in their entireties). To clinically harness the potential therapeutic benefits of butyrate esters to treat allergic and inflammatory disorders of the lower gastrointestinal (GI) tract (e.g., food allergies, inflammatory bowel disease, etc.), more controlled and practical delivery strategies are needed.

[0007] Short-chain fatty acid therapeutics have been difficult to translate into clinical use because they degrade rapidly during passage through the intestine, and the molecules themselves, both as free bases and acid salts, have an unpleasant taste, a foul odor, and cause stomach upset. Although enteric-coated short-chain fatty acid products are commercially available, they are not widely used, in part due to the drawbacks mentioned above and the inability to densely pack sufficient amounts of pharmaceutically active short-chain fatty acids to exert a therapeutic effect.

[0008] A delivery system that overcomes the above limitations would be useful for all known clinical uses of the short chain fatty acids listed above. Summary of the Invention

[0009] Provided herein are prodrug polymeric micelles that find use in the delivery of short chain fatty acids to the intestine for promoting gut health, establishing a healthy microbiota, and treating immune and / or inflammatory diseases such as inflammatory bowel disease and food allergies.

[0010] Provided herein are copolymers (e.g., random or block) that are delivery vehicles for short- to medium-chain hydrophobic or amphiphilic carboxylic acids (e.g., 3-12 carbon atoms in the chain, collectively referred to herein as short-chain fatty acids, "SCFAs") and functionalized derivatives of these acids. In some embodiments, the copolymers are delivery vehicles for butyrate esters. The polymers provide delivery of the SCFAs to the intestine, including the mucosal lining of the small and large intestine, and in certain embodiments, the ileum and cecum. The SCFAs and / or their derivatives are covalently attached to the copolymer backbone, which is hydrolytically or enzymatically cleavable, thereby releasing the SCFAs and providing the desired therapeutic effect for human diseases. The therapeutic effect targets the barrier function of the intestine and intestinal mucus layer, potentially treating any disease associated with mucus layer thickness or barrier function. In some embodiments, the therapeutic effect is the promotion of gut health, the establishment or maintenance of a healthy gut flora (e.g., Clostridium species), the treatment of inflammatory diseases (e.g., IBD), and / or the treatment of immune diseases (e.g., food allergies). Exemplary human diseases treatable with the polymers described herein include, but are not limited to, autoimmune diseases (e.g., rheumatoid arthritis, celiac disease), allergic and atopic diseases (e.g., all types of food allergies, eosinophilic esophagitis, allergic rhinitis, allergic asthma, pet allergies, drug allergies), inflammatory diseases (e.g., inflammatory bowel disease, ulcerative colitis, Crohn's disease), infectious diseases, metabolic disorders, diseases of the central nervous system (e.g., multiple sclerosis, Alzheimer's disease, Parkinson's disease), blood disorders (e.g., beta-thalassemia), colon cancer, diseases affecting gut motility (e.g., diarrhea), type 1 diabetes, and autism spectrum disorders, among others. The copolymers may be administered by any convenient route (eg, oral administration, rectal administration, etc.) and overcome known limitations associated with the administration of short chain fatty acids (eg, butyrate esters) alone.

[0011] Embodiments herein relate to copolymers (e.g., random or block) of (i) a monomer comprising MMA and (ii) a monomer exposing an SCFA moiety (e.g., butyrate ester) and attached to the copolymer by a methacrylate or methacrylamide group, their supramolecular assemblies (e.g., micelles), nanoparticles comprising such copolymers, and methods of using them. In some embodiments, the copolymers comprise two types of monomers in a random or pseudo-random distribution. In another embodiment, the copolymer is a block copolymer comprising an MAA block and a block containing a monomer that exposes an SCFA moiety (e.g., a butyrate ester) and is bonded to the copolymer by a methacrylate ester group or a methacrylamide group (e.g., a poly(N-oxyethyl methacrylate) block that exposes an SFCA, a poly(N-oxyethyl methacrylamide) block that exposes an SFCA, a poly(N-(4-hydroxybenzoyloxy)alkyl methacrylamide) block that exposes an SFCA, a poly(N-(4-hydroxybenzoyloxy)alkyl methacrylate) block that exposes an SFCA, etc.).

[0012] Advantages of the polymeric drug delivery systems described herein, in which pharmaceutically active SCFAs (e.g., butyrate esters) are covalently attached to the polymer chains, include masking the odor of the SCFAs, improving their palatability, and increasing their bioavailability, especially in the distal intestine, where other forms are unsuitable for therapeutic use. In some embodiments, micelles comprising the copolymers described herein are provided. In some embodiments, SCFA-loaded micelles can also be more densely packed and deliver therapeutically relevant doses of bioactive molecules. In some embodiments, the delivery systems described herein survive gastric passage and, upon hydrolysis caused by pH changes or enzymatic cleavage, e.g., by bacterial or host esterases, deliver therapeutically required payloads of SCFAs to the intestinal barrier, thus serving as attractive options for short-chain fatty acid delivery.

[0013] In some embodiments, provided herein are copolymers (e.g., block or random) of (i) MAA monomers and (ii) N-oxyalkylmethacrylamide monomers (or poly(N-oxyalkylmethacrylamide) blocks) having SCFA moieties (e.g., butyrate esters) or other pharmaceutically suitable small molecules covalently attached to the blocks.

[0014] In some embodiments, the N-oxyalkyl methacrylamide monomer (or poly(N-oxyalkyl methacrylamide) block) comprises a monomer selected from the group consisting of oxymethyl methacrylamide, 2-oxyethyl methacrylamide, 3-oxypropyl methacrylamide, N-oxyisopropyl methacrylamide, 4-oxybutyl methacrylamide, N-oxyisobutyl methacrylamide, or an N-oxyalkyl methacrylamide having a longer or otherwise branched or substituted alkyl chain. In some embodiments, the N-oxyalkyl methacrylamide (or poly(N-oxyalkyl methacrylamide) block) comprises a linear alkyl chain of 1 to 20 carbons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range therebetween, e.g., 2 to 8). In some embodiments, the N-oxyalkyl methacrylamide (or poly(N-oxyalkyl methacrylamide) block) comprises a branched alkyl group of 1 to 20 carbons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range therebetween, e.g., 2-8), such as 2-methylpentyl, 3-ethylpentyl, 3,3-dimethylhexyl, 2,3-dimethylhexyl, 4-ethyl-2-methylhexyl, or any other suitable branched alkyl group. In some embodiments, the N-oxyalkyl methacrylamide (or poly(N-oxyalkyl methacrylamide) block) comprises one or more double or triple carbon-carbon bonds (e.g., alkenyl or alkynyl instead of alkanyl). In some embodiments, the N-oxyalkyl methacrylamide (or poly(N-oxyalkyl methacrylamide) block) comprises a heteroalkyl group (e.g., (CH)) that includes one of the alkyl groups described above (e.g., straight or branched chain) having one or more heteroatoms (e.g., O, S, NH, etc.) replacing one of the carbons in the alkyl group. n X(CH2) mwherein m and n are independently 1 to 10, and X is O, S, or NH. In some embodiments, the N-oxyalkyl methacrylamide (or poly(N-oxyalkyl methacrylamide) block) comprises a substituted alkyl group, including one of the alkyl groups described above (e.g., linear or branched chain) having one or more pendant substituents (e.g., OH, NH, ═O, halogen (e.g., Cl, F, Br, I), CN, CF, etc.). In some embodiments, the poly(N-oxyalkyl methacrylamide) comprises a linear or branched chain alkyl group containing any and appropriate combination of heteroatoms, pendant substituents, double bonds, etc. In certain embodiments, the N-oxyalkyl methacrylamide (or poly(N-oxyalkyl methacrylamide) block) is a 2-oxyalkyl methacrylamide (or poly(2-oxyalkyl methacrylamide) block).

[0015] In some embodiments, provided herein are copolymers (e.g., block or random) of (i) MAA monomers and (ii) N-oxyalkylphenol ester methacrylamides (or poly(N-oxyalkylphenol ester methacrylamides) blocks) having SCFA moieties (e.g., butyrate esters) or other pharmaceutically suitable small molecules covalently attached to the blocks.

[0016] In some embodiments, the N-oxyalkyl 4-phenol ester methacrylamide monomer (or poly(N-oxyalkyl 4-phenol ester methacrylamide) block) comprises a monomer selected from the group consisting of oxymethyl 4-phenol methacrylamide, 2-oxyethyl 4-phenol methacrylamide, 3-oxypropyl 4-phenol methacrylamide, 4-oxybutyl 4-phenol methacrylamide, or an N-oxyalkyl 4-phenol ester methacrylamide having a longer or otherwise branched or substituted alkyl chain. In some embodiments, the N-oxyalkyl 4-phenol ester methacrylamide monomer (or poly(N-oxyalkyl 4-phenol ester methacrylamide) block) comprises a linear alkyl chain of 1 to 20 carbons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range therebetween, e.g., 2 to 8). In some embodiments, the N-oxyalkyl 4-phenol ester methacrylamide monomer (or poly(N-oxyalkyl 4-phenol ester methacrylamide) block) comprises a branched alkyl group of 1 to 20 carbons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range therebetween, e.g., 2 to 8), such as 2-methylpentyl, 3-ethylpentyl, 3,3-dimethylhexyl, 2,3-dimethylhexyl, 4-ethyl-2-methylhexyl, or any other suitable branched alkyl group. In some embodiments, the N-oxyalkyl 4-phenol ester methacrylamide monomer (or poly(N-oxyalkyl 4-phenol ester methacrylamide) block) comprises one or more double or triple carbon-carbon bonds (e.g., alkenyl or alkynyl instead of alkanyl).In some embodiments, the N-oxyalkyl 4-phenol ester methacrylamide monomer (or poly(N-oxyalkyl 4-phenol ester methacrylamide) block) comprises a heteroalkyl group (e.g., (CH2)), which comprises one of the alkyl groups described above (e.g., linear or branched) having one or more heteroatoms (e.g., O, S, NH, etc.) replacing one of the carbons in the alkyl group. n X(CH2) m wherein m and n are independently 1 to 10, and X is O, S, or NH. In some embodiments, the N-oxyalkyl 4-phenol ester methacrylamide monomer (or poly(N-oxyalkyl 4-phenol ester methacrylamide) block) comprises a substituted alkyl group, including one of the alkyl groups described above (e.g., linear or branched chain) having one or more pendant substituents (e.g., OH, NH, ═O, halogen, (e.g., Cl, F, Br, I), CN, CF, etc.). In some embodiments, the poly(N-oxyalkyl methacrylamide) comprises a linear or branched alkyl group containing any and appropriate combination of heteroatoms, pendant substituents, double bonds, etc. In certain embodiments, the N-oxyalkyl 4-phenol methacrylamide is poly(2-oxyethyl 4-phenol methacrylamide).

[0017] In some embodiments, the poly(N-oxyalkyl 4-phenolmethacrylamide), with or without the alkyl modifications described above, is substituted at any position on the phenol ring with a moiety selected from groups including, but not limited to, alkyl, hydroxyl, alkoxyl, amine, N-alkylamine, carboxyl, halogen, nitro, and derivatives thereof.

[0018] In some embodiments, provided herein are copolymers (e.g., block or random) of (i) MAA monomers and (ii) N-oxyalkyl methacrylate ester monomers (or poly(N-oxyalkyl methacrylate) blocks) having SCFA moieties or other pharmaceutically suitable small molecules covalently attached to the blocks.

[0019] In some embodiments, the N-oxyalkyl methacrylate monomer (or poly(N-oxyalkyl methacrylate) block) comprises a monomer selected from the group consisting of oxymethyl methacrylate, 2-oxyethyl methacrylate, 3-oxypropyl methacrylate, N-oxyisopropyl methacrylate, 4-oxybutyl methacrylate, N-oxyisobutyl methacrylate, or an N-oxyalkyl methacrylate with a longer or otherwise branched or substituted alkyl chain. In some embodiments, the N-oxyalkyl methacrylate (or poly(N-oxyalkyl methacrylate) block) comprises a linear alkyl chain of 1 to 20 carbons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or ranges therebetween, e.g., 2 to 8). In some embodiments, the N-oxyalkyl methacrylate (or poly(N-oxyalkyl methacrylate) block) comprises a branched alkyl group of 1 to 20 carbons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or ranges therebetween, e.g., 2-8), such as 2-methylpentyl, 3-ethylpentyl, 3,3-dimethylhexyl, 2,3-dimethylhexyl, 4-ethyl-2-methylhexyl, or any other suitable branched alkyl group. In some embodiments, the N-oxyalkyl methacrylate (or poly(N-oxyalkyl methacrylate) block) comprises one or more double or triple carbon-carbon bonds (e.g., alkenyl or alkynyl instead of alkanyl). In some embodiments, the N-oxyalkyl methacrylate (or poly(N-oxyalkyl methacrylate) block) comprises a heteroalkyl group (e.g., (CH)) that includes one of the alkyl groups described above (e.g., straight or branched chain) having one or more heteroatoms (e.g., O, S, NH, etc.) replacing one of the carbons in the alkyl group. n X(CH2) mwherein m and n are independently 1 to 10, and X is O, S, or NH. In some embodiments, the N-oxyalkyl methacrylate (or poly(N-oxyalkyl methacrylate) block) comprises a substituted alkyl group including one of the above-mentioned alkyl groups (e.g., linear or branched) having one or more pendant substituents (e.g., OH, NH, ═O, halogen, (e.g., Cl, F, Br, I), CN, CF, etc.). In some embodiments, the poly(N-oxyalkyl methacrylate) comprises a linear or branched alkyl group containing any and appropriate combination of heteroatoms, pendant substituents, double bonds, etc. In certain embodiments, the N-oxyalkyl methacrylate (or poly(N-oxyalkyl methacrylate) block) is a 2-oxyalkyl methacrylate (or poly(2-oxyalkyl methacrylate) block).

[0020] In some embodiments, provided herein are copolymers (e.g., block or random) of (i) an MAA monomer or block and (ii) a methacrylic acid N-oxyalkylphenol ester (or a poly(methacrylic acid N-oxyalkylphenol ester) block) having an SCFA moiety or other pharmaceutically suitable small molecule covalently attached to the block.

[0021] In some embodiments, the N-oxyalkyl 4-phenol methacrylate ester monomer (or poly(N-oxyalkyl 4-phenol methacrylate ester) block) comprises a monomer selected from the group consisting of oxymethyl 4-phenol methacrylate, 2-oxyethyl 4-phenol methacrylate, 3-oxypropyl 4-phenol methacrylate, 4-oxybutyl 4-phenol methacrylate, or an N-oxyalkyl 4-phenol methacrylate with a longer or otherwise branched or substituted alkyl chain. In some embodiments, the N-oxyalkyl 4-phenol methacrylate ester monomer (or poly(N-oxyalkyl 4-phenol methacrylate ester) block) comprises a linear alkyl chain of 1 to 20 carbons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range therebetween, e.g., 2 to 8). In some embodiments, the methacrylic acid N-oxyalkyl 4-phenol ester monomer (or poly(methacrylic acid N-oxyalkyl 4-phenol ester) block) comprises a branched alkyl group of 1 to 20 carbons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range therebetween, e.g., 2-8), such as 2-methylpentyl, 3-ethylpentyl, 3,3-dimethylhexyl, 2,3-dimethylhexyl, 4-ethyl-2-methylhexyl, or any other suitable branched alkyl group. In some embodiments, the methacrylic acid N-oxyalkyl 4-phenol ester monomer (or poly(methacrylic acid N-oxyalkyl 4-phenol ester) block) comprises one or more double or triple carbon-carbon bonds (e.g., alkenyl or alkynyl instead of alkanyl).In some embodiments, the methacrylic acid N-oxyalkyl 4-phenol ester monomer (or poly(methacrylic acid N-oxyalkyl 4-phenol ester) block) comprises a heteroalkyl group (e.g., (CH2)) that includes one of the alkyl groups described above (e.g., linear or branched) having one or more heteroatoms (e.g., O, S, NH, etc.) replacing one of the carbons in the alkyl group. n X(CH2) m where m and n are independently 1 to 10, and X is O, S, or NH. In some embodiments, the N-oxyalkyl 4-phenol methacrylate ester monomer (or poly(N-oxyalkyl 4-phenol methacrylate ester) block) comprises a substituted alkyl group comprising one of the above-mentioned alkyl groups (e.g., linear or branched) having one or more pendant substituents (e.g., OH, NH, ═O, halogen, (e.g., Cl, F, Br, I), CN, CF, etc.). In some embodiments, the poly(N-oxyalkyl methacrylate) comprises a linear or branched alkyl group containing any and appropriate combination of heteroatoms, pendant substituents, double bonds, etc. In certain embodiments, the N-oxyalkyl 4-phenol methacrylate is poly(2-oxyethyl 4-phenol methacrylate).

[0022] In some embodiments, the poly(N-oxyalkyl 4-phenol methacrylate), with or without the alkyl modifications described above, is substituted at any position on the phenol ring with a moiety selected from groups including, but not limited to, alkyl, hydroxyl, alkoxyl, amine, N-alkylamine, carboxyl, halogen, nitro, and derivatives thereof.

[0023] In some embodiments, the block comprises a polymer of MAA monomers. In certain embodiments, the MAA block is poly(MAA). In certain embodiments, the molecular weight of the polyMAA block is 7,000 to 15,000 Da (e.g., 7,000 Da, 8,000 Da, 9,000 Da, 10,000 Da, 11,000 Da, 12,000 Da, 13,000 Da, 14,000 Da, 15,000 Da, or a range therebetween (e.g., 9,000 to 14,000 Da)).

[0024] In certain embodiments, the copolymer comprises a covalently attached SCFA moiety or other pharmaceutically suitable small molecule. In some embodiments, the SCFA moiety is selected from the group consisting of acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, caprylic acid, capric acid, lauric acid, and derivatives thereof. In some embodiments, any fatty acid having an aliphatic tail of 12 or fewer carbons (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or any range therein (e.g., 3-10)) can be used in embodiments herein. In certain embodiments, the SCFA moiety is a butyrate ester (butyric acid) or an isobutyrate ester (isobutyric acid).

[0025] In some embodiments, the ratio of the MAA block to the SCFA-exposing (or other pharmaceutically suitable small molecule-exposing) block is between 0.25 and 3.5 (e.g., 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, or a range therebetween (e.g., 0.7-1.8)). In some embodiments, the ratio of MAA monomers to SCFA-exposing (or other pharmaceutically suitable small molecule-exposing) monomers is between 0.5 and 2.0 (e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or ranges therebetween (e.g., 0.7-1.8)).

[0026] In some embodiments, provided herein are polymers comprising an incorporation ratio of MAA to SCFA-exposing monomers of 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20 (or any range therebetween).

[0027] In some embodiments, the polymer comprises 20-80 weight percent (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or any range therebetween) of MAA monomers. In some embodiments, the polymer comprises 20-80 weight percent (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or any range therebetween) of SCFA-exposing monomers.

[0028] In some embodiments, provided herein are copolymers comprising an MAA monomer (or polyMAA block) and an SCFA moiety (e.g., butyrate or isobutyrate ester) or other pharmaceutically suitable small molecule covalently attached to the copolymer via a linker group by a methacrylate or methacrylamide group.

[0029] In some embodiments, provided herein are supramolecular assemblies (e.g., micelles) comprising a plurality of copolymers described herein (e.g., comprising SCFAs or other small molecule cargoes) (e.g., dispersed in a liquid). In some embodiments, the assemblies are nanoparticles having diameters of 10-1000 nm (e.g., 10, 20, 50, 100, 200, 500, 1000 nm, or ranges therebetween, e.g., 50-500 nm). In certain embodiments, the plurality of block copolymers comprises linear and branched copolymers that self-assemble or covalently bond to form nanoparticles. In other embodiments, the assemblies are micelles. In still other embodiments, the supramolecular assemblies (e.g., micelles) are isolated (e.g., as a powder) and redispersed (e.g., in a liquid).

[0030] In some embodiments, provided herein are methods for delivering a target molecule (e.g., an SCFA) to a subject (e.g., a human subject, a male subject, a female subject, etc.), the method comprising: providing a supramolecular assembly (e.g., a micelle) of a copolymer described herein comprising the target molecule (e.g., an SCFA); and contacting the supramolecular assembly (e.g., a micelle) with the subject, thereby delivering the target molecule to the subject. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising the block copolymer described herein and / or a supramolecular assembly (e.g., a micelle) thereof is administered to a subject by any suitable administration route. In some embodiments, the target molecule (e.g., an SCFA) is covalently bound to the supramolecular assembly (e.g., a micelle). In certain embodiments, the supramolecular assembly (e.g., a micelle) is orally contacted (e.g., administered) to a subject. In some embodiments, the supramolecular assembly (e.g., a micelle) is dispersed in a liquid carrier when contacted with the subject. In other embodiments, the supramolecular assembly (e.g., a micelle) is solid when contacted with the subject. In some embodiments, the supramolecular assembly (eg, micelle) is for use as a pharmaceutical.

[0031] In some embodiments, provided herein is the use of supramolecular assemblies (eg, micelles) of the copolymers described herein in the manufacture of a pharmaceutical product.

[0032] In some embodiments, provided herein is a pharmaceutical composition comprising the supramolecular assembly (e.g., micelle) described herein. In certain embodiments, the supramolecular assembly (e.g., micelle) is combined with a pharmaceutically acceptable (e.g., considered safe and effective) carrier and administered to a subject (e.g., without causing undesired biological side effects or undesired interactions).

[0033] In some embodiments, described herein is a monomer comprising (i) methacrylic acid (MAA) and (ii) a monomer of formula (I): [ka] wherein X is O, NH, or S, and L is a linker selected from an alkyl chain, a heteroalkyl chain, a substituted alkyl chain, or a substituted heteroalkyl chain, wherein the copolymer has one or more exposed short chain fatty acid (SCFA) moieties.

[0034] In some embodiments, described herein is a monomer comprising (i) methacrylic acid (MAA) and (ii) a monomer of formula (II): [ka] Compositions are provided that include copolymers with monomers of formula (II), wherein X is O, NH, or S; L is a linker selected from an alkyl chain, a heteroalkyl chain, a substituted alkyl chain, or a substituted heteroalkyl chain; and SCFA is a short chain fatty acid.

[0035] In some embodiments, L in Formula (I) or Formula (II) is (CH) n wherein n is 1 to 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a range therebetween). In some embodiments, L is (CH2). n O(CO)-benzene. In some embodiments, the SCFA is covalently bound to the monomer of formula (I). In some embodiments, the SCFA bound to the monomer of formula (I) comprises a monomer of formula (II). [ka] In some embodiments, the SCFA attached to the monomer of formula (I) or the monomer of formula (II) is of formula (III): [ka] In some embodiments, the SCFA is selected from the group consisting of acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, caprylic acid, capric acid, lauric acid, branched chain forms thereof, and derivatives thereof. In some embodiments, the SCFA is butyric acid.

[0036] In some embodiments, the copolymer is a block copolymer comprising an MAA block and a block of formula (I) or (II). In some embodiments, the block copolymer is a block copolymer comprising a block of formula (IV): [ka] wherein M h contains MAA, and M F2 is the side chain of the monomer of formula (II), [ka] a is 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or a range therebetween), and b is 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or a range therebetween).

[0037] In some embodiments, the copolymer is a random copolymer. In some embodiments, the random copolymer comprises formula (V): [ka] wherein each Y independently represents a side chain of a polymer formed from formula (II): [ka] and The side chain of MAA, [ka] In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0038] In some embodiments, the monomer of Formula (II) comprises N-butanoyloxyalkyl methacrylamide. In some embodiments, the N-butanoyloxyalkyl methacrylamide monomer is 2-butanoyloxyethyl methacrylamide. In some embodiments, the copolymer is a block copolymer and comprises Formula (VI): [ka] wherein a and b are independently 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or ranges therebetween). In some embodiments, the copolymer is a random copolymer and comprises formula (V): [ka] wherein each Y independently represents (i) the side chain of MAA; [ka] and (ii) side chains of poly(2-butanoyloxyethyl methacrylamide). In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0039] In some embodiments, the monomer of Formula (II) comprises N-butanoyloxyalkyl methacrylate. In some embodiments, the N-butanoyloxyalkyl methacrylate monomer is 2-butanoyloxyethyl methacrylate. In some embodiments, the copolymer is a block copolymer and comprises Formula (VII): [ka] wherein a and b are independently 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or ranges therebetween). In some embodiments, the copolymer is a random copolymer and comprises formula (V): [ka] wherein each Y independently represents (i) the side chain of MAA; [ka] and (ii) the side chains of poly(2-butanoyloxyethyl methacrylate). In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0040] In some embodiments, the monomer of formula (II) comprises an N-(4-butanoyloxybenzoyloxy)alkyl methacrylate. In some embodiments, the N-(4-butanoyloxybenzoyloxy)alkyl methacrylate monomer is 2-(4-butanoyloxybenzoyloxy)ethyl methacrylate. In some embodiments, the copolymer is a block copolymer and comprises formula (VIII): [ka] wherein a and b are independently 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or ranges therebetween). In some embodiments, the copolymer is a random copolymer and comprises formula (V): [ka] wherein each Y independently represents (i) the side chain of MAA; [ka] and (ii) side chains of poly(2-(4-butanoyloxybenzoyloxy)ethyl methacrylate), [ka] In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0041] In some embodiments, the monomer of formula (II) comprises N-(4-butanoyloxybenzoyloxy)alkyl methacrylamide. In some embodiments, the N-(4-butanoyloxybenzoyloxy)alkyl methacrylamide monomer is 2-(4-butanoyloxybenzoyloxy)ethyl methacrylamide. In some embodiments, the copolymer is a block copolymer and comprises formula (IX): [ka] wherein a and b are independently 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or ranges therebetween). In some embodiments, the copolymer is a random copolymer and comprises formula (V): [ka] wherein each Y independently represents (i) the side chain of MAA; [ka] and (ii) side chains of poly(2-(4-butanoyloxybenzoyloxy)ethyl methacrylamide); [ka] In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0042] In some embodiments, the compositions herein comprise a second copolymer or micelle thereof (e.g., in addition to the MAA-containing copolymer), the second copolymer comprising (i) a monomer comprising N-(2-hydroxyethyl) methacrylamide (HPMA) and (ii) a monomer of formula (II)2: [ka] wherein X2 is O, NH, or S, L2 is a linker selected from an alkyl chain, a heteroalkyl chain, a substituted alkyl chain, or a substituted heteroalkyl chain, and SCFA2 is a short chain fatty acid. In some embodiments, L2 is (CH2) n wherein n is 1 to 16. In some embodiments, L2 is (CH2) n In some embodiments, the monomer of formula (II)2 is formula (III)2: [ka]

[0043] In some embodiments, the SCFA2 is selected from the group consisting of acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, caprylic acid, capric acid, lauric acid, branched chain forms thereof, and derivatives thereof. In some embodiments, the SCFA2 is butyric acid. In some embodiments, the second copolymer comprises 10 to 80% by weight of butyric acid. In some embodiments, the second copolymer is a block copolymer comprising an HPMA block and a block of formula (II)2. In some embodiments, the second copolymer is a random copolymer. For example, in some embodiments, the second copolymer comprises formula (V)2, [ka] wherein each Y2 independently represents a side chain of formula (II)2, [ka] and Selected from the side chains of polyHPMA [ka] In some embodiments, the monomer of Formula (II)2 comprises N-butanoyloxyalkyl methacrylamide, hi some embodiments, the N-butanoyloxyalkyl methacrylamide monomer is 2-butanoyloxyethyl methacrylamide.

[0044] In some embodiments, the second copolymer is a block copolymer and comprises Formula (VI): [ka] In the formula, a and b are independently 1 to 1000. In some embodiments, the second copolymer is a random copolymer and comprises formula (V)2: [ka] wherein each Y2 independently represents (i) a side chain of polyHPMA; [ka] and (ii) The side chain is selected from poly(2-butanoyloxyethyl methacrylamide). [ka]

[0045] In some embodiments, the monomer of Formula (II)2 comprises N-butanoyloxyalkyl methacrylate. In some embodiments, the N-butanoyloxyalkyl methacrylate monomer is 2-butanoyloxyethyl methacrylate. In some embodiments, the second copolymer is a block copolymer and comprises Formula (VII)2: [ka] In the formula, a and b are independently 1 to 1000. In some embodiments, the second copolymer is a random copolymer and comprises formula (V)2: [ka] wherein each Y2 independently represents (i) a side chain of polyHPMA; [ka] and (ii) 2-butanoyloxyethyl methacrylate. In some embodiments, the monomer of Formula (II)2 comprises an N-(4-butanoyloxybenzoyloxy)alkyl methacrylate monomer. In some embodiments, the N-(4-butanoyloxybenzoyloxy)alkyl methacrylate monomer is 2-(4-butanoyloxybenzoyloxy)ethyl methacrylate. In some embodiments, the second copolymer is a block copolymer and comprises Formula (VIII)2: [ka] In the formula, a and b are independently 1 to 1000. In some embodiments, the second copolymer is a random copolymer and comprises formula (V)2: [ka] wherein each Y2 independently represents (i) a side chain of polyHPMA; [ka] and (ii) The side chains of poly(2-(4-butanoyloxybenzoyloxy)ethyl methacrylate) are selected. [ka]

[0046] In some embodiments, the monomer of Formula (II)2 comprises an N-(4-butanoyloxybenzoyloxy)alkyl methacrylamide monomer. In some embodiments, the N-(4-butanoyloxybenzoyloxy)alkyl methacrylamide monomer is 2-(4-butanoyloxybenzoyloxy)ethyl methacrylamide. In some embodiments, the second copolymer is a block copolymer and comprises Formula (IX)2: [ka] In the formula, a and b are independently 1 to 1000. In some embodiments, the second copolymer is a random copolymer and comprises formula (V)2: [ka] wherein each Y2 independently represents (i) a side chain of polyHPMA; [ka] and (ii) The side chain is selected from poly(2-(4-butanoyloxybenzoyloxy)ethyl methacrylamide). [ka]

[0047] In some embodiments, provided herein are compositions comprising: (a) poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-alkanoyloxyethyl) methacrylamide) (pHPMA-b-pAMA); and (b) poly(methacrylic acid)-b-poly(N-(2-alkanoyloxyethyl) methacrylamide) (pMAA-b-pAMA). In some embodiments, the pMAA-b-pAMA exists as a negatively charged micelle. In some embodiments, the pHPMA-b-pAMA exists as a neutrally charged micelle. In some embodiments, the pMAA-b-pAMA exists as poly(methacrylic acid)-b-poly(N-(2-methanoyloxyethyl) methacrylamide). (pMAA-b-pMMA), poly(methacrylic acid)-b-poly(N-(2-alkanoyloxyethyl)methacrylamide) (pMAA-b-pEMA), poly(methacrylic acid)-b-poly(N-(2-propanoyloxyethyl)methacrylamide) (pMAA-b-pPMA), poly(methacrylic acid)-b-poly(N-(2-butanoyloxyethyl)methacrylamide) (pMAA-b-pBMA), poly(methacrylic acid)-b-poly(N-(2-pentanoyloxyethyl)methacrylamide) (pMAA-b-pPeMA), poly(methacrylic acid)-b-poly(N-(2-hexanoyloxyethyl)methacrylamide) (pMAA-b-pHMA), or longer SCFA-containing copolymers.In some embodiments, the pHPMA-b-pAMA is selected from the group consisting of poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-methanoyloxyethyl) methacrylamide) (pHPMA-b-pMMA), poly(2-hydroxypropyl ethacrylamide)-b-poly(N-(2-alkanoyloxyethyl) methacrylamide) (pHPMA-b-pEMA), poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-propanoyloxyethyl) methacrylamide) (pHPMA- b-pPMA), poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-butanoyloxyethyl)methacrylamide) (pHPMA-b-pBMA), poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-pentanoyloxyethyl)methacrylamide) (pHPMA-b-pPeMA), poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-hexanoyloxyethyl)methacrylamide) (pHPMA-b-pHMA), or longer SCFA-containing copolymers. In some embodiments, provided herein is a pharmaceutical composition comprising a composition described herein and a pharmaceutically acceptable carrier. In some embodiments, provided herein is a food or nutraceutical composition comprising a composition described herein and an edible carrier. In some embodiments, provided herein is a method comprising administering a pharmaceutical composition, food, or nutraceutical composition described herein to a subject in need thereof. In some embodiments, the subject suffers from a food allergy. In some embodiments, the subject suffers from a dysbiosis. In some embodiments, the subject is receiving an antibiotic. In some embodiments, the method results in an increase in the abundance and / or relative abundance of Enterococcus, Coprobacter, and Clostridium cluster XIVa. In some embodiments, the method results in an increase in the abundance and / or relative abundance of bacteria of the family Lachnospiraceae.In some embodiments, the methods result in an increase in the abundance and / or relative abundance of Clostridium cluster XIVa, IV, and / or XVIII bacteria. In some embodiments, the methods result in improved intestinal barrier function, reduced inflammation, improved physician scores, improved patient-reported outcomes, and / or reduced sensitivity to allergens. In some embodiments, the methods result in increased production of butyrate esters and other beneficial metabolites by the subject's gut microbiota.

[0048] In some embodiments, provided herein are methods of establishing a healthy gut microbiota in a subject, the method comprising administering a composition comprising (a) poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-alkanoyloxyethyl) methacrylamide) (pHPMA-b-pAMA), or (b) poly(methacrylic acid)-b-poly(N-(2-alkanoyloxyethyl) methacrylamide) (pMAA-b-pAMA) to a subject in need thereof. In some embodiments, the pHPMA-b-pAMA is selected from the group consisting of poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-methanoyloxyethyl) methacrylamide) (pHPMA-b-pMMA), poly(2-hydroxypropyl ethacrylamide) (ethacrylamido), and poly(methacrylic acid). poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-alkanoyloxyethyl)methacrylamide) (pHPMA-b-pEMA), poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-propanoyloxyethyl)methacrylamide) (pHPMA-b-pPMA), poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-butanoyloxyethyl)methacrylamide) (pHPMA-b-pBMA), poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-pentanoyloxyethyl)methacrylamide) (pHPMA-b-pPeMA), poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-hexanoyloxyethyl)methacrylamide) (pHPMA-b-pHMA), or longer SCFA-containing copolymers.In some embodiments, the pMAA-b-pAMA is selected from the group consisting of poly(methacrylic acid)-b-poly(N-(2-methanoyloxyethyl)methacrylamide) (pMAA-b-pMMA), poly(methacrylic acid)-b-poly(N-(2-alkanoyloxyethyl)methacrylamide) (pMAA-b-pEMA), poly(methacrylic acid)-b-poly(N-(2-propanoyloxyethyl)methacrylamide) (pMAA-b-pPMA), The method comprises one or more of poly(methacrylic acid)-b-poly(N-(2-butanoyloxyethyl)methacrylamide) (pMAA-b-pBMA), poly(methacrylic acid)-b-poly(N-(2-pentanoyloxyethyl)methacrylamide) (pMAA-b-pPeMA), poly(methacrylic acid)-b-poly(N-(2-hexanoyloxyethyl)methacrylamide) (pMAA-b-pHMA), or longer SCFA-containing copolymers. In some embodiments, the subject suffers from a dysbiosis. In some embodiments, the subject is receiving an antibiotic. In some embodiments, the method results in an increase in the relative abundance of Enterococcus, Coprobacter, and Clostridium cluster XIVa. In some embodiments, the method results in an increase in the abundance and / or relative abundance of bacteria of the family Lachnospiraceae. In some embodiments, the methods result in an increase in the abundance and / or relative abundance of Clostridium cluster XIVa, IV, and / or XVIII bacteria. In some embodiments, the methods result in improved intestinal barrier function, reduced inflammation, improved physician scores, improved patient-reported outcomes, and / or reduced sensitivity to allergens. In some embodiments, the methods result in increased production of butyrate esters and other beneficial metabolites by the subject's gut microbiota.

[0049] In some embodiments, provided herein are methods that include: (a) detecting bacteria and / or bacterial metabolites in the stool of a subject; and (b) administering to the subject a composition that includes (i) poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-alkanoyloxyethyl) methacrylamide) (pHPMA-b-pAMA), or (ii) poly(methacrylic acid)-b-poly(N-(2-alkanoyloxyethyl) methacrylamide) (pMAA-b-pAMA). In some embodiments, the pHPMA-b-pAMA is selected from the group consisting of poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-methanoyloxyethyl) methacrylamide) (pHPMA-b-pMMA), poly(2-hydroxypropyl ethacrylamide)- and one or more of b-poly(N-(2-alkanoyloxyethyl)methacrylamide) (pHPMA-b-pEMA), poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-propanoyloxyethyl)methacrylamide) (pHPMA-b-pPMA), poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-butanoyloxyethyl)methacrylamide) (pHPMA-b-pBMA), poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-pentanoyloxyethyl)methacrylamide) (pHPMA-b-pPeMA), poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-hexanoyloxyethyl)methacrylamide) (pHPMA-b-pHMA), or longer SCFA-containing copolymers.In some embodiments, the pMAA-b-pAMA is selected from the group consisting of poly(methacrylic acid)-b-poly(N-(2-methanoyloxyethyl)methacrylamide) (pMAA-b-pMMA), poly(methacrylic acid)-b-poly(N-(2-alkanoyloxyethyl)methacrylamide) (pMAA-b-pEMA), poly(methacrylic acid)-b-poly(N-(2-propanoyloxyethyl)methacrylamide) (pMAA-b-pPMA), The composition comprises one or more of poly(methacrylic acid)-b-poly(N-(2-butanoyloxyethyl)methacrylamide) (pMAA-b-pBMA), poly(methacrylic acid)-b-poly(N-(2-pentanoyloxyethyl)methacrylamide) (pMAA-b-pPeMA), poly(methacrylic acid)-b-poly(N-(2-hexanoyloxyethyl)methacrylamide) (pMAA-b-pHMA), or longer SCFA-containing copolymers. In some embodiments, the composition is administered if the subject is determined to be suffering from a dysbiosis or intestinal metabolite deficiency. In some embodiments, the detecting is performed before and / or after administration of the composition. In some embodiments, the detecting is used to determine whether continued administration of the composition would be beneficial to the subject. In some embodiments, the detecting is used to determine an appropriate dose of the composition.

[0050] In some embodiments, provided herein are methods comprising: (a) administering to a subject a first dose of a composition comprising (i) poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-alkanoyloxyethyl) methacrylamide) (pHPMA-b-pAMA), and / or (ii) poly(methacrylic acid)-b-poly(N-(2-alkanoyloxyethyl) methacrylamide) (pMAA-b-pAMA); and (b) administering to the subject a second, lower dose of the composition. In some embodiments, the pHPMA-b-pAMA is selected from the group consisting of poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-methanoyloxyethyl) methacrylamide) (pHPMA-b-pMMA), poly(2-hydroxypropyl ethacrylamide) (ethacrylamidomethyl) methyl acrylate (methacrylate), and / or poly(methacrylic acid). poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-alkanoyloxyethyl)methacrylamide) (pHPMA-b-pEMA), poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-propanoyloxyethyl)methacrylamide) (pHPMA-b-pPMA), poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-butanoyloxyethyl)methacrylamide) (pHPMA-b-pBMA), poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-pentanoyloxyethyl)methacrylamide) (pHPMA-b-pPeMA), poly(2-hydroxypropylmethacrylamide)-b-poly(N-(2-hexanoyloxyethyl)methacrylamide) (pHPMA-b-pHMA), or longer SCFA-containing copolymers.In some embodiments, the pMAA-b-pAMA is selected from the group consisting of poly(methacrylic acid)-b-poly(N-(2-methanoyloxyethyl)methacrylamide) (pMAA-b-pMMA), poly(methacrylic acid)-b-poly(N-(2-alkanoyloxyethyl)methacrylamide) (pMAA-b-pEMA), poly(methacrylic acid)-b-poly(N-(2-propanoyloxyethyl)methacrylamide) (pMAA-b-pPMA), The compositions include one or more of poly(methacrylic acid)-b-poly(N-(2-butanoyloxyethyl)methacrylamide) (pMAA-b-pBMA), poly(methacrylic acid)-b-poly(N-(2-pentanoyloxyethyl)methacrylamide) (pMAA-b-pPeMA), poly(methacrylic acid)-b-poly(N-(2-hexanoyloxyethyl)methacrylamide) (pMAA-b-pHMA), or longer SCFA-containing copolymers. In some embodiments, a first dose is administered multiple times over a first time span, followed by a second, lower dose. In some embodiments, the first dose is administered twice daily, once daily, or once weekly for a first time span. In some embodiments, the first time span is one (1) week, two (2) weeks, three (3) weeks, one (1) month, two (2) months, four (4) months, six (6) months, one (1) year, or more, or any range therebetween. In some embodiments, the first dose is 1 to 40 g (e.g., 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 21 g, 22 g, 23 g, 24 g, 25 g, 26 g, 27 g, 28 g, 29 g, 30 g, 31 g, 32 g, 33 g, 34 g, 35 g, 36 g, 37 g, 38 g, 39 g, 40 g, 41 g, 42 g, 43 g, 44 g, 45 g, 46 g, 47 g, 48 g, 49 g, 50 g, 51 g, 52 g, 53 g, 54 g, 55 g, 56 g, 57 g, 58 g, 59 g, 60 g, 61 g, 62 g, 63 g, 64 g, 65 g, 66 g, 67 g, 68 g, 69 g, 70 g, 71 g, 72 g, 73 g, 74 g, 75 g, 76 g, 77 g, 78 g, 79 g, 80 g, 81 g, 82 g, 83 g, 84 g, 85 g, 86 g, 7 g, 38 g, 39 g, 40 g, or a range therebetween) of (i) poly(2-hydroxypropyl methacrylamide)-b-poly(N-(2-butanoyloxyethyl) methacrylamide (pHPMA-b-pBMA), and / or (ii) poly(methacrylic acid)-b-poly(N-(2-butanoyloxyethyl) methacrylamide (pMAA-b-pBMA).In some embodiments, the second, lower dose is between one-tenth (1 / 10) and one-half (1 / 2) of the first dose (e.g., 0.1×, 0.2×, 0.3×, 0.4×, 0.5×, or a range therebetween). In some embodiments, step (b) is performed after performing step (a) for a predetermined time span (e.g., 1 week, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months). In some embodiments, step (b) is performed after assessing the subject's gut microbiota. In some embodiments, the subject suffered from dysbiosis before step (a). In some embodiments, step (b) is performed after assessing the level of one or more gut metabolites in the subject. In some embodiments, the one or more gut metabolites comprise a short-chain fatty acid. In some embodiments, the short-chain fatty acid comprises a butyrate ester. In some embodiments, the method further comprises one or more steps of assessing the subject's gut microbiota and / or assessing the level of one or more gut metabolites in the subject prior to step (a), between steps (a) and (b), and / or after step (b).

[0051] In some embodiments, provided herein are compositions comprising first micelles of a first copolymer of methacrylic acid (MAA) and N-(2-alkanoyloxyethyl) methacrylamide (AMA). In some embodiments, the copolymer is a block copolymer having the following structure: [ka] wherein a and b are independently 1 to 1000. In some embodiments, the copolymer is a random copolymer having the following structure: [ka] wherein each Y independently represents: [ka] In some embodiments, the copolymer is a block copolymer having the structure: [ka] wherein a and b are independently 1 to 1000. In some embodiments, the copolymer is a random copolymer having the following structure: [ka] wherein each Y independently represents: [ka]

[0052] In some embodiments, the copolymer is a block copolymer having the structure: [ka] wherein a and b are independently 1 to 1000. In some embodiments, the copolymer is a random copolymer having the following structure: [ka] wherein each Y independently represents: [ka]

[0053] In some embodiments, the copolymer is a block copolymer and has the structure: [ka] wherein a and b are independently 1 to 1000. In some embodiments, the copolymer is a random copolymer and has the following structure: [ka] wherein each Y independently represents: [ka] In some embodiments, the copolymer is a block copolymer having the structure: [ka] wherein a and b are independently 1 to 1000. In some embodiments, the copolymer is a random copolymer and has the following structure: [ka] wherein each Y independently represents: [ka]

[0054] In some embodiments, the copolymer is a block copolymer having the structure: [ka] wherein a and b are independently 1 to 1000. In some embodiments, the copolymer is a random copolymer having the following structure: [ka] wherein each Y independently represents: [ka]

[0055] In some embodiments, the composition further comprises a second micelle of a second copolymer of 2-hydroxypropyl methacrylamide (HPMA) and N-(2-alkanoyloxyethyl) methacrylamide (AMA). In some embodiments, the second copolymer is a block copolymer having the following structure: [ka] In the formula, a and b are independently 1 to 1000. In some embodiments, the second copolymer is a random copolymer having the following structure: [ka] wherein each Y independently represents: [ka]

[0056] In some embodiments, the second copolymer is a block copolymer having the structure: [ka] In the formula, a and b are independently 1 to 1000. In some embodiments, the second copolymer is a random copolymer having the following structure: [ka] wherein each Y independently represents: [ka]

[0057] In some embodiments, the second copolymer is a block copolymer having the structure: [ka] In the formula, a and b are independently 1 to 1,000.

[0058] In some embodiments, the second copolymer is a random copolymer having the structure: [ka] wherein each Y independently represents: [ka]

[0059] In some embodiments, the second copolymer is a block copolymer and has the structure: [ka] In the formula, a and b are independently 1 to 1,000.

[0060] In some embodiments, the second copolymer is a random copolymer and has the structure: [ka] wherein each Y independently represents: [ka]

[0061] In some embodiments, the second copolymer is a block copolymer having the structure: [ka] wherein a and b are independently 1 to 1000. In some embodiments, the second copolymer is a random copolymer and has the following structure: [ka] wherein each Y independently represents: [ka]

[0062] In some embodiments, the second copolymer is a block copolymer having the structure: [ka] In the formula, a and b are independently 1 to 1000. In some embodiments, the second copolymer is a random copolymer having the following structure: [ka] wherein each Y independently represents: [ka]

[0063] In some embodiments, provided herein are supramolecular assemblies (e.g., micelles) of the copolymers described herein. In some embodiments, the supramolecular assemblies are micelles or nanoparticles. In some embodiments, provided herein are compositions comprising two or more different types of supramolecular assemblies (e.g., micelles), for example, comprising different copolymers (e.g., MAA-AMA and HPMA-AMA copolymers).

[0064] In some embodiments, provided herein are pharmaceutical compositions comprising a supramolecular assembly (e.g., a micelle) or copolymer described herein and a pharmaceutically acceptable carrier.

[0065] In some embodiments, provided herein are food or nutraceutical compositions comprising supramolecular assemblies (eg, micelles) or copolymers described herein.

[0066] In some embodiments, provided herein are methods comprising administering to a subject a pharmaceutical composition, food, or nutraceutical composition described herein. In some embodiments, the methods are performed to treat or prevent a disease or condition. In some embodiments, the disease or condition is selected from the group consisting of an autoimmune disease, an allergy, an inflammatory condition, an infection, a metabolic disorder, a disease of the central nervous system, colon cancer, diabetes, and autism spectrum disorder.

[0067] In some embodiments, provided herein are methods of synthesizing or producing the copolymers, supramolecular assemblies (e.g., micelles), pharmaceutical compositions, foods, and / or nutraceutical compositions described herein.

[0068] In some embodiments, provided herein are uses of the copolymers, supramolecular assemblies (e.g., micelles), pharmaceutical compositions, foods, and / or nutraceutical compositions described herein for the treatment or prevention of a disease or disorder. [Brief explanation of the drawings]

[0069] [Figure 1] The 1H-NMR (500 MHz, CDCl3) of HEMA (2) is shown. [Figure 2] The 1H-NMR (500 MHz, CDCl3) of BMA (3) is shown. [Figure 3] 1H-NMR (500 MHz, DMSO-d6) of pMAA(7) is shown. [Figure 4] 1H-NMR (500 MHz, DMSO-d6) of pMAA-b-pBMA (8) is shown. [Figure 5]Chemical composition and structural characteristics of butyrate-prodrug micelles, i.e., NtL-ButM, composed of the neutral block copolymer pHPMA-b-pBMA, and Neg-ButM, composed of the anionic block copolymer pMAA-b-pBMA, are shown. A, Synthetic route for pHPMA-b-pBMA and pMAA-b-pBMA. B. (Top) NtL-ButM contains a hydrophilic (HPMA) block as the micelle corona, while the hydrophobic (BMA) block forms the micelle core. (Bottom) Neg-ButM contains a hydrophilic (MAA) block that forms a negatively charged micelle corona, and the same hydrophobic (BMA) block as NtL-ButM. C, D, Cryo-electron microscopy (cryoEM) images show the spherical structure of the micelles NtL-ButM (C) or Neg-ButM (D). E, Table summarizing the characteristics of micelles NtL-ButM and Neg-ButM, including hydrodynamic diameter and zeta potential from DLS, critical micelle concentration, radius of gyration, and aggregation number from SAXS. [Figure 6] A, Both NtL-ButM and Neg-ButM slowly released butyrate ester in simulated gastric fluid over 20 days. B, Both NtL-ButM and Neg-ButM released their full butyrate ester load within minutes in simulated intestinal fluid (SIF) containing high levels of the esterase pancreatin. Neither polymer released butyrate ester in PBS over these time scales. n=3. [Figure 7] Biodistribution of NtL-ButM or Neg-ButM in the gastrointestinal tract (GI) as measured by in vivo imaging system (IVIS). Both polymers were chemically modified with azide and labeled with the dye IR750. IVIS showed that NtL-ButM rapidly translocated to the cecum after a single oral administration to mice, while Neg-ButM remained in the stomach for more than 6 hours. Both polymers were excreted from the GI tract after 24 hours. [Figure 8]The amount of butyrate esters released into the ileum, cecum, or colon contents after a single intragastric administration of 0.8 mg / g NtL-ButM or Neg-ButM to SPF C3H / HeJ mice is shown. Butyrate esters were derivatized with 3-nitrophenylhydrazine and quantified by LC-MS / MS (ileum samples) or LC-UV (cecum and colon samples). n = 9–10 mice per group. Data represent the mean ± SEM. [Figure 9] Figure 1 shows the tissue and cellular biodistribution of butyrate-releasing polymers. A. Representative IVIS images of lymph nodes from mice injected sc with fluorescently labeled NtL-ButM or Neg-ButM in the abdomen. Quantification of fluorescent signal from different tissues 3 or 7 days after sc injection. n = 3 mice per group. C. Percentage of fluorescent NtL-ButM or Neg-ButM positive cells among different cell subsets in inguinal LNs, small intestinal draining LNs, or spleen. n = 6 mice per group per time point. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. [Figure 10] CD40 and CD86 expression on mouse APCs in draining LNs during s.c. LPS stimulation after s.c. administration of butyrate micelles. n=5 mice per group. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.005. [Figure 11]A. C57B / 6 WT Foxp3GFP+ mice received antibiotic water or regular water throughout the experiment and were treated weekly with either PBS, NtL-ButM, or Neg-ButM for 3 weeks, starting 3 days after weaning. Mice were sacrificed 1 week after the final treatment, and the Treg population in different tissues was analyzed by flow cytometry and the amount of butyrate esters by LC-MS. B. Percentage of Treg (CD25+GFP+) among CD4+ T cells (CD45+CD3+CD4+) in different LNs and spleens of mice. C. Percentage of RoRγt+ cells among Treg (CD45+CD3+CD4+Foxp3+CD25+) in different LNs and spleens of mice. D. Butyrate ester analysis in liver, spleen, serum, and colon contents from different groups of mice. n = 3–6 mice per group. Data represent mean ± SEM. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. [Figure 12] Chemical composition and structural characteristics of butyrate-prodrug micelles, i.e., NtL-ButM, composed of the neutral block copolymer pHPMA-b-pBMA, and Neg-ButM, composed of the anionic block copolymer pMAA-b-pBMA. a) Synthetic routes for pHPMA-b-pBMA and pMA-b-pBMA. b) (Top) NtL-ButM contains a hydrophilic (HPMA) block as the micelle corona, while the hydrophobic (BMA) block forms the micelle core. (Bottom) Neg-ButM contains a hydrophilic (MAA) block that forms a negatively charged micelle corona, and the same hydrophobic (BMA) block as NtL-ButM. c) and d) Cryo-electron microscopy (cryoEM) images show the spherical structure of the micelles NtL-ButM (c) and Neg-ButM (d). e, Table summarizing the characteristics of micelles NtL-ButM and Neg-ButM, including hydrodynamic diameter and zeta potential from DLS, critical micelle concentration, radius of gyration, and aggregation number from SAXS. [Figure 13]In vitro and in vivo butyrate release in the GI tract from NtL-ButM and Neg-ButM are shown. a) Both NtL-ButM and Neg-ButM slowly released butyrate in simulated gastric fluid over a 20-day period. b) Both NtL-ButM and Neg-ButM released their full butyrate load within minutes in simulated intestinal fluid (SIF) containing high levels of the esterase pancreatin. Neither polymer released butyrate in PBS over these timescales. n = 3. c-e) Amount of butyrate released into the ileum, cecum, or colon contents after a single intragastric administration of NtL-ButM or Neg-ButM at 0.8 mg / g to SPF C3H / HeJ mice. Butyrate was derivatized with 3-nitrophenylhydrazine and quantified by LC-MS / MS (ileum samples) or LC-UV (cecum and colon samples). The dotted red line represents the butyrate content in untreated mice. n = 9–10 mice per group. Data represent the mean ± sem. [Figure 14] NtL-ButM induced an ileal gene expression signature that was almost entirely antimicrobial peptide (AMP). a) Daily administration of 0.8 mg / g NtL-ButM to germ-free (GF) C3H / HeN mice for 1 week induced a unique gene expression signature in the ileum compared with untreated and inactive polymer controls, as measured by RNA sequencing of isolated intestinal epithelial cells. The top 100 significantly differentially expressed genes (DEGs) with false discovery rate (FDR)-adjusted P < 0.005 and fold change (FC) ≥ 1.5 or ≤ -1.5 are shown. Annotation bars for the three groups, experimental batches (E2 and E3), and sex (female, male) are indicated above the heatmap. b) Fluorescence imaging of intelectin protein in small intestinal sections from control or treated mice. Blue (DAPI), red (intelectin). c) Intelectin protein is quantified by total fluorescent signal per small intestinal crypt. >15 crypts were quantified per mouse, n = 3 PBS-treated and 4 NtL-ButM-treated mice. Data represent mean ± sem. Precision weighted limma volume was used in a. Two-tailed Student's t-test was used in c. ***P < 0.001. [Figure 15] Butyrate micelle treatment restored intestinal barrier integrity in DSS- or antibiotic-treated mice. a) Epithelial barrier dysfunction was induced in mice by administering 2.5% DSS in drinking water for 7 days. DSS was removed from drinking water on days 7–10. For treatment, mice were intragastrically administered with either PBS, different concentrations of cyclosporine A (CsA), or ButM daily. QD: once daily; BID: twice daily at 10–12 h intervals. On day 10, all mice received an ig dose of 4 kDa FITC-dextran. Fluorescence was measured in serum 4 h later. b) Concentration of FITC-dextran in serum. n = 8 mice per group, except for the high-dose ButM group, which had 16 mice per group. c) Mice were treated with a mixture of antibiotics for 7 days starting at 2 weeks of age. After weaning, mice received 800 mg / kg of either PBS (n = 10) or ButM (n = 11) intravenously twice daily for 7 days. All mice then received an intravenous dose of 4 kDa FITC-dextran. Fluorescence was measured in serum 1.5 hours later. d, Concentration of FITC-dextran in serum. Data in d are pooled from two independent experiments. Data represent mean ± sem. Comparisons were made using one-way ANOVA with Dunnett's post-hoc test (c) or Student's t-test (d). *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 16]Butyrate micelle treatment reduced the anaphylactic response to peanut challenge. a, b. Experimental scheme and administration strategy. All mice were sensitized weekly by gavage with 6 mg of peanut extract (PN) plus 10 mg of the mucosal adjuvant cholera toxin. Four weeks after sensitization, one group of mice (n = 20) was challenged with 1 mg of PN administered ip to confirm that the sensitization protocol induced a uniform allergic response. Fecal samples were collected before and after treatment for microbiota analysis in Figure 6. (c) Changes in core body temperature after PN challenge, where a drop in core body temperature indicates anaphylaxis. The remaining mice were randomized into two treatment groups. One group was treated with PBS (n = 32), and the other group was treated with a 1:1 mixture of NtL-ButM and Neg-ButM polymers (0.4 mg / g each) (n = 41). QD: once daily, BID: twice daily at 10-12 hour intervals. d, Changes in core body temperature after challenge with PN in PBS- or ButM-treated mice. Area under the curve (AUC) values ​​were compared between the two groups. e, f, Serum mMCPT-1 (e) and peanut-specific IgE (f) from mice in d. Data represent mean ± sem. Data in c, d, and e are pooled from two independent experiments. Data were analyzed using a two-tailed Student's t-test. *P<0.05, ***P<0.001. [Figure 17] Butyrate micelles alter the fecal microbiota after antibiotic exposure, promoting the recovery of Clostridium cluster XIVa. a) 16S rRNA sequencing analysis of the relative abundance of bacterial taxa in fecal samples from allergic mice collected before (left) or after (right) treatment with PBS (n = 8) or ButM (n = 17) (see Figure 5a). b) Differentially abundant taxa between PBS- or ButM-treated mice after treatment, as analyzed by LEfSec. c) Relative abundance of Clostridium cluster XIVa in fecal samples after treatment with PBS or ButM (from a), or d) analyzed by qPCR. For c and d, a Student's t-test with Welch's correction was used for statistical analysis. **P < 0.01. [Figure 18]The 1H-NMR (500 MHz, CDCl3) of HEMA (2) is shown. [Figure 19] The 1H-NMR (500 MHz, CDCl3) of BMA (3) is shown. [Figure 20] 1H-NMR (500 MHz, DMSO-d6) of pHPMA (5) is shown. [Figure 21] 1H-NMR (500 MHz, DMSO-d6) of pHPMA-b-pBMA (6) is shown. [Figure 22] 1H-NMR (500 MHz, DMSO-d6) of pMAA(7) is shown. [Figure 23] 1H-NMR (500 MHz, DMSO-d6) of pMAA-b-pBMA (8) is shown. [Figure 24] The H-NMR (500 MHz, CDCl) of N3-PEG4-MA (9) is shown. [Figure 25] 1H-NMR (500 MHz, CDCl3) of N-hexylmethacrylamide (10) is shown. [Figure 26] 1H-NMR (500 MHz, DMSO-d6) of the control polymer pHPMA-b-pHMA is shown. [Figure 27] Dynamic light scattering (DLS) shows that the micelles NtL-ButM and Neg-ButM have similar hydrodynamic diameters of less than 100 nanometers. [Figure 28] The critical micelle concentrations (CMCs) of NtL-ButM (left) and Neg-ButM (right) are shown, as measured by pyrene fluorescence intensity of peak 1 relative to peak 3. The CMCs were determined by IC50 fitted by a sigmoidal curve. [Figure 29]Small-angle X-ray scattering (SAXS) characterization of NtL-ButM and Neg-ButM micelles. a, SAXS data for NtL-ButM and Neg-ButM. Data are fitted to a polydisperse core-shell model. b, Gunier plot (ln(q) vs. q²) of NtL-ButM reveals the radius of gyration of the micelles. c, Kratky plot (lq² vs. q²) of NtL-ButM reveals the spherical structure of the micelles. d, Gunier plot of Neg-ButM micelles. e, Kratky plot of Neg-ButM micelles. f, Table of fitting parameters for NtL-ButM and Neg-ButM using a polydisperse core-shell spherical model. g, Table of average intermicelle distances. d, Number of micelles per unit volume N, molecular weight of the micelle Mw, and aggregation number Nagg calculated from the fitting parameters of the polydisperse core-shell spherical model. [Figure 30] Derivatization of butyrate esters for LC-MS / MS analysis and release of butyrate esters from NtL-ButM / Neg-ButM into simulated gastric / intestinal fluid. a) Derivatization of butyrate esters with 3-nitrophenylhydrazine (NPH) to generate UV-active butyrate ester-NPH. b) Quantification of butyrate ester-NPH in LC-MS / MS using multiple reaction monitoring (MRM) of 222→137. [Figure 31]Stability of pHPMA-b-pBMA polymer in vitro or in vivo. a) Gel permeation chromatography (GPC) elution profile (measured by differential refractive index (dRI) over time) of polymer collected from pooled fecal samples of two mice treated with NtL-ButM after 4–6 h (red) or 6–8 h (blue) of gavage. Black curve: polymer control. b) Table of molecular weight of digested polymer measured by GPC, including number-average molecular weight (Mn) and weight-average molecular weight (Mw), along with polydispersity index (PDI) and Mn loss, compared to undigested polymer control. c, d) Mn loss of pHPMA-b-pBMA polymer in 125 mM NaOH solution over 7 days (c) or percentage of butyrate released from the polymer measured by GPC (d). n = 3. Data represent mean ± s.e.m. [Figure 32] Biodistribution of NtL-ButM or Neg-ButM in the gastrointestinal (GI) tract (a), as well as other major organs and serum (b), as measured by in vivo imaging system (IVIS). Both polymers were chemically modified with azide and labeled with the dye IR750. IVIS showed that after a single oral dose of NtL-ButM or Neg-ButM (one mouse per treatment group per time point), Neg-ButM remained in the stomach for more than 6 hours, whereas NtL-ButM rapidly translocated to the cecum after a single intragastric administration to mice. Both polymers were excreted from the GI tract after 24 hours, and none of the butyrate micelles were absorbed into the systemic circulation. Mesenteric LN (d, duodenal inflow area; j, jejunal inflow area; I, ileal inflow area; c, colonic inflow area). [Figure 33] Figure 3 shows differentially expressed genes (DEGs) in the ileum of GF mice treated with 0.8 mg / g NtL-ButM daily for 1 week compared to untreated and inactive polymer controls, as measured by RNA sequencing of isolated ileal epithelial cells. Values ​​are in TMM-normalized, log2-transformed read counts. [Figure 34]Butyrate micelle treatment reduced the anaphylactic response to peanut challenge in a dose-dependent manner. a, b, Experimental scheme and administration strategy. All mice were sensitized weekly by ig gavage of 6 mg PN + 10 μg mucosal adjuvant cholera toxin. c, One group of mice (n = 7) was challenged with 1 mg PN ip, and a uniform allergic response was confirmed by measuring a decrease in core body temperature as an indicator of anaphylaxis. QD: once daily. d-f, The remaining mice were randomized into three treatment groups and received either PBS (n = 8), 0.4 mg / g (half dose) ButM (n = 11), or 0.8 mg / g (full dose) ButM (n = 9) twice daily. d, Changes in core body temperature after challenge with peanut extract. The area under the curve (AUC) was compared between groups. Serum mMCPT-1 (e) and peanut-specific IgE (f) from mice in e, f, and d. Data represent mean ± sem. Data analyzed using one-way ANOVA with Dunnett's post-hoc test. *P<0.05. [Figure 35]Butyrate micelle treatment combined with low-dose PN reduced the anaphylactic response to peanut challenge. a, b, Experimental scheme and administration strategy. All mice were sensitized weekly by ig gavage with 6 mg PN + 10 μg of mucosal adjuvant cholera toxin. c, One group of mice (n = 46) was challenged with 1 mg PN ip, and a uniform allergic response was confirmed by measuring a decrease in core body temperature as an indicator of anaphylaxis. QD: once daily. d, The remaining mice were randomized into two treatment groups. One group (n = 26) was treated once daily with low-dose PB2 (200 μg, blue), and the other group (n = 29) was treated once daily with low-dose PB2 200 μg + 0.8 mg / g ButM twice daily. d, Changes in core body temperature after challenge with peanut extract in low-dose PN- or low-dose PN + ButM-treated mice. The area under the curve (AUC) was compared between the two groups. Serum mMCPT-1 (e) and peanut-specific IgE (f) from mice in e, f, d. Data represent the mean ± sem. Data were analyzed using Student's t-test. **P<0.01. ns, not significant. [Figure 36] Differentially abundant taxa within each treatment group, as analyzed by LEfSe from Figure 6 , before and after 2 weeks of treatment with PBS (a) or ButM (b). [Figure 37] NtL-ButM showed no serological toxicity in mice. SPF C3H / HeJ mice were treated daily for 6 weeks with PBS, sodium butyrate (NaBut), or NtL-ButM. Mouse serum samples were measured weekly for six toxicity markers using a chemistry analyzer. As an example, the results for alanine aminotransferase (ALT) levels at week 6 are shown in a. b. None of the markers showed significant differences between the NtL-ButM and PBS groups. Data represent mean ± sem. Comparisons were performed using one-way ANOVA with Dunnett's post-hoc test. ns, no significant difference.

[0070] Definition of Terms Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, certain preferred methods, compositions, devices, and materials are described herein. However, before the materials and methods are described, it should be understood that the invention is not limited to the particular molecules, compositions, methodologies, or protocols described herein, as these may vary according to routine experimentation and optimization. It should also be understood that the terminology used in the description is for the purpose of describing particular aspects or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0072] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "block copolymer" is a reference to one or more block copolymers and equivalents thereof known to those skilled in the art, and so forth.

[0073] As used herein, the term "comprise" and its linguistic variations indicate the presence of the stated feature(s), element(s), method step(s), etc., without excluding the presence of additional feature(s), element(s), method step(s), etc. In contrast, the term "consisting of" and its linguistic variations indicate the presence of the stated feature(s), element(s), method step(s), etc., but excludes any unrecited feature(s), element(s), method step(s), etc., except for impurities normally associated therewith. The phrase "consisting essentially of" indicates the stated feature(s), element(s), method step(s), etc., as well as any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open "comprising" language. Such embodiments encompass multiple closed "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such language.

[0074] As used herein, the term "short chain fatty acid" ("SCFA") refers to a carboxylic acid attached to a saturated or unsaturated aliphatic chain, the aliphatic chain being 12 carbons or less in length.

[0075] As used herein, the term "fatty acid derivative" (particularly "SCFA derivative") refers to a small molecule compound obtained by simple modification (e.g., amidation, methylation, halogenation, etc.) of a fatty acid molecule (e.g., an SCFA molecule). For example, butyramido, D- or L-amino-n-butyric acid, α- or β-amino-n-butyric acid, arginine butyrate, butyrin, phenylbutyrate (e.g., 4-, 3-, 2-), dimethylbutyrate, 4-halobutyrate (e.g., fluoro-, chloro-, bromo-, iodo-), 3-halobutyrate (e.g., fluoro-, chloro-, bromo-, iodo-), 2-halobutyrate (e.g., fluoro-, chloro-, bromo-, iodo-), oxybutyrate, and methylbutyrate are exemplary butyrate derivatives. Other butyrate derivatives and similar derivatives of other SCFAs are within the scope of the SCFA derivatives described herein.

[0076] As used herein, the term "copolymer" refers to a polymer formed from two or more different monomer subunits. Exemplary copolymers include alternating copolymers, random copolymers, block copolymers, etc.

[0077] As used herein, the term "block copolymer" refers to a copolymer in which the repeating subunits are polymer blocks, i.e., polymers of polymers. In a copolymer of block A and block B, A and B each represent the polymeric entity obtained by polymerization of the monomers. Exemplary configurations of such block copolymers include branched, star, diblock, triblock, etc.

[0078] As used herein, the term "supramolecule" (e.g., "supramolecular assembly" (e.g., micelle)) refers to non-covalent interactions between molecules and / or solutions (e.g., polymers, macromolecules, etc.) and the resulting multi-component assemblies, complexes, systems, and / or fibers. In some embodiments, micelles are supramolecular assemblies that result from non-covalent interactions between copolymers, for example, in colloidal solutions.

[0079] As used herein, the term "dysbiosis" refers to a decrease in microbial diversity, including an increase in pathogenic bacteria and / or a loss of beneficial bacteria, such as Bacteroides strains, Enterococcus, Coprobacter, and Clostridium cluster XIVa bacteria, as well as other butyrate-producing bacteria, such as Firmicutes.

[0080] As used herein, the term "abundance," when used in reference to bacteria, refers to the amount of bacteria of a certain type present. The term "relative abundance," when used in reference to bacteria, refers to the amount of bacteria of a certain type present compared to the overall amount of bacteria present.

[0081] As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, excipient, or carrier conventionally used for use with a therapeutic agent for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation employed. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is administered orally, the carrier may be a gel capsule. A "pharmaceutical composition" generally comprises at least one active agent (e.g., a copolymer described herein) and a pharmaceutically acceptable carrier.

[0082] As used herein, the term "effective amount" refers to the amount of a composition (e.g., a pharmaceutical composition) sufficient to achieve a beneficial or desired result. An effective amount may be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0083] As used herein, the term "administration" refers to the act of providing a drug, prodrug, or other agent or therapeutic treatment (e.g., a pharmaceutical composition of the present invention) to a subject or to cells, tissues, and organs in vivo, in vitro, or ex vivo. Exemplary routes of administration to the human body can be via the eye (e.g., intraocular, intravitreal, periocular, ophthalmic, etc.), mouth (oral), skin (transdermal), nose (intranasal), lung (inhalation), oral mucosa (buccal), ear, rectum, or by injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.).

[0084] As used herein, the terms "combination" and "combined" refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the combination of two or more agents or therapies is simultaneous (e.g., in the same or separate formulations). In other embodiments, a first agent / therapy is administered before a second agent / therapy. Those of skill in the art will appreciate that the formulations and / or routes of administration of the various agents or therapies used may vary. Appropriate dosages for combinations can be readily determined by those of skill in the art. In some embodiments, when agents or therapies are combined, each agent or therapy is administered at a lower dosage than would be appropriate for its administration alone. Thus, combinations are particularly desirable in embodiments where the combination of agents or therapies reduces the required dosage of a potentially harmful (e.g., toxic) agent(s).

[0085] As used herein, the term "nanoparticle" refers to a particle having an average dimension (e.g., diameter, width, length, etc.) of less than 1 μm (e.g., <500 nm ("sub-500 nm nanoparticles"), <100 nm ("sub-100 nm nanoparticles"), <50 nm ("sub-50 nm" nanoparticles").

[0086] As used herein, the term "biocompatible" refers to a material, compound, or composition that does not cause or induce significant adverse effects when administered to a subject. Examples of adverse effects that may limit biocompatibility include, but are not limited to, excessive inflammation, excessive or adverse immune responses, and toxicity.

[0087] As used herein, the term "biostable" refers to a composition or material that does not readily degrade or degrade in a physiological or similar aqueous environment. Conversely, as used herein, the term "biodegradable" refers to a composition or material that readily degrades (e.g., depolymerizes, hydrolyzes, enzymatically degrades, dissociates, etc.) in a physiological or other environment.

[0088] As used herein, the term "substituted" refers to a group (such as alkyl) modified with one or more additional groups. Non-limiting examples of substituents include, for example, halogen, hydroxy, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO), imino (=NH), oximo (=N-OH), hydrazino (=N-NH), NH-R. b- OR a , -R b- OC(O)-R a , -R b- OC(O)-OR a , -R b- OC(O)-N(R a )2, -R b- N(R a )2, -R b- C(O)R a , -R b- C(O)OR a , -R b- C(O)N(R a )2, -R b- OR c- C(O)N(R a )2, -R b- N(R a )C(O)OR a , -R b- N(R a )C(O)R a , -R b- N(R a )S(O) t R a (t is 1 or 2), -R b- S(O) t R a (t is 1 or 2), -R b- S(O) t OR a(t is 1 or 2), and -R b- S(O) t N(R a )2, where t is 1 or 2; and each optionally is halogen, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R b- OR a , -R b- OC(O)-R a , -R b- OC(O)-OR a , -R b- OC(O)-N(R a )2, -R b- N(R a )2, -R b- C(O)R a , -R b- C(O)OR a , -R b- C(O)N(R a )2, -R b- OR c- C(O)N(R a )2, -R b- N(R a )C(O)OR a , -R b- N(R a )C(O)R a , -R b- N(R a )S(O) t R a (t is 1 or 2), -R b- S(O) t R a (t is 1 or 2), -R b- S(O) t OR a (t is 1 or 2), -R b- S(O) t N(R a ) 2 (t is 1 or 2), carbocycle, and heterocycle, and optionally substituted alkyl, alkenyl, and alkynyl, wherein each R a are independently selected from hydrogen, alkyl, alkenyl, alkynyl, carbocycle, and heterocycle; and each R ais optionally, where valence allows, halogen, oxo (=O), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=NH), oximo (=N-OH), hydrazine (=N-NH2), -R b- OR a , -R b- OC(O)-R a , -R b- OC(O)-OR a , -R b- OC(O)-N(R a )2, -R b- N(R a )2, -R b- C(O)R a , -R b- C(O)OR a , -R b- C(O)N(R a )2, -R b- OR c- C(O)N(R a )2, -R b- N(R a )C(O)OR a , -R b- N(R a )C(O)R a , -R b- N(R a )S(O) t R a (t is 1 or 2), -R b- S(O) t R a (t is 1 or 2), -R b- S(O) t OR a (t is 1 or 2), and -R b- S(O) t N(R a )2 (t is 1 or 2), and each R b is independently selected from a direct bond or a straight or branched chain alkylene, alkenylene, or alkynylene chain; and each R cis a straight or branched alkylene, alkenylene, or alkynylene chain. The substituents can be selected from, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, hydroxyl, alkoxy, mercaptyl, cyano, halo, carbonyl, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, and amino (including mono- and di-substituted amino groups), and protected derivatives thereof. "Substituted alkyl" encompasses alkynes and alkenes in addition to the alkane that represents the substituent moiety.

[0089] As used herein, the term "pseudorandom" refers to sequences or structures produced by a process in which no steps or measures are taken to control the order of addition of monomers or components.

[0090] As used herein, the term "expose" refers to the presentation of a solvent-exposed functional group by a molecule, monomer, polymer, nanostructure, or other chemical entity. DETAILED DESCRIPTION OF THE INVENTION

[0091] Provided herein are polymeric materials that find use, for example, in the delivery of short-chain fatty acids. In particular, provided are polymers that form stable nanoscale structures (e.g., micelles) and release their payload, for example, by cleavage of covalent bonds (e.g., by hydrolysis or enzymatic cleavage). The polymers are useful, for example, for delivering payloads (e.g., short-chain fatty acids (SCFAs)) to the intestine for health and disease treatment applications, and have broad applicability to diseases associated with alterations in the human microbiota, including inflammatory diseases, autoimmune diseases, allergic diseases, metabolic diseases, and central nervous system diseases, among others. In some embodiments, provided herein are prodrug polymer micelles that find use in delivering short-chain fatty acids to the intestine for promoting gut health, establishing a healthy microbiota, and treating immune and / or inflammatory diseases such as inflammatory bowel disease and food allergies.

[0092] During the development of embodiments herein, experiments were conducted to develop block copolymers capable of loading a high content of butyrate esters into the core of water-suspendable micelles. These polymer formulations mask the odor and taste of butyrate esters and act as carriers to release active ingredients (e.g., SCFAs (e.g., butyrate esters)) over time as the micelles pass through the GI tract. Experiments conducted during the development of embodiments herein have shown that these butyrate ester-conjugated polymer formulations upregulate AMP gene expression in the ileal epithelium and modulate barrier integrity in antibiotic-treated mice and mice treated with dextran sulfate sodium (DSS), a chemical perturbant that induces epithelial barrier dysfunction. Intragastric administration of the butyrate ester-prodrug micelles of the present invention ameliorated the anaphylactic response to peanut challenge and increased the abundance of bacteria in a cluster known to contain butyrate ester-producing taxa (Clostridium cluster XIVa) in a mouse model of peanut allergy.

[0093] The prevalence of food allergies has increased dramatically over the past two decades, particularly in developed countries (References B7, B46; incorporated by reference in their entirety). Lifestyle changes, such as decreased dietary fiber intake, increased antibiotic use (including in the food chain), and sanitation, have altered the population of commensal microorganisms. These changes result in several negative health effects, including impaired intestinal barrier function. Modulating the gut microbiota to redirect immunity has become a significant effort in both academia and industry. However, this has proven challenging, and colonizing the gut with selected organisms, particularly anaerobic bacteria, is not straightforward.

[0094] Described herein is a polymeric nanoscale system for delivering SCFAs (e.g., butyrate esters) to localized regions along the GI tract. This system is based on polymeric micelles formed by block copolymers, in which SCFAs (e.g., butyrate esters) are conjugated to hydrophobic blocks via ester bonds, allowing them to be hydrolyzed by esterases in the GI tract for localized release. The linked butyrate ester moiety drives the hydrophobicity within the block, and upon release, the remainder of the construct (the inert, water-soluble polymer) continues to travel through the lower GI tract until excretion. The butyrate ester-containing block, when forming the core of the micelles, is resistant to the acidic environment found in the stomach, preventing burst release in the stomach before the micelles migrate to the intestine. Two butyrate ester-prodrug micelles, NtL-ButM and Neg-ButM, share a similar structure but possess neutral and negative corona charges, respectively. This allows for the release of butyrate esters in the lower GI tract in the presence of enzymes, resulting in a unique biodistribution in the lower GI tract. During the development of embodiments herein, we conducted experiments using both neutrally and negatively charged micelles to deliver butyrate ester along the distal intestine to treat a mouse model of peanut allergy and repair intestinal barrier dysfunction. Experiments demonstrated successful preservation of barrier function and protection from severe anaphylactic responses with short-term treatment. By inducing a gene expression signature composed almost entirely of AMPs, butyrate ester micelles are able to act locally without absorption in the small intestine. These AMPs are primarily expressed by specialized Paneth cells in the ileum and are essential for maintaining the balance of the ileal microbiota (see Reference B15; incorporated by reference in its entirety). In a mouse model of peanut allergy in which mice had previously been exposed to vancomycin to induce dysbiosis, ButM treatment favorably increased the relative abundance of protective bacteria, such as Clostridium cluster XIVa.Bacteria of Clostridium cluster XIVa are known to induce local Tregs in preclinical models and may be important for the success of fecal microbiota transplantation for the treatment of colitis (References B43, B47; incorporated by reference in their entireties).

[0095] Investigating the therapeutic potential of butyrate esters in animal models, butyrate esters were supplemented in drinking water or diet at high doses for periods of three weeks or more (see References B16-B18, B23-B25; incorporated by reference in their entireties). Such administration to achieve a therapeutic effect from sodium butyrate is difficult to translate clinically due to uncontrolled dosing regimens, difficulty in replicating in humans, and the unpleasant odor and taste of butyrate esters as sodium salts. The use of formulations, such as those used during the development of embodiments herein, incorporating butyrate esters into polymeric micelles at high loadings (28% by weight), is capable of delivering and releasing a majority of butyrate esters in the lower GI tract in a manner that masks the taste and odor of butyrate esters. Peanut-allergic mice were treated for two weeks using a daily dose of 800 mg / kg total ButM. This can be converted to a human dose of approximately 65 mg / kg total ButM (or an equivalent butyrate dose of 18.2 mg / kg), taking into account the difference in body surface area between rodents and humans (Reference B48; incorporated by reference in its entirety). This butyrate dose in ButM micelles is comparable to other clinically tested butyrate formulations (References B49-B51; incorporated by reference in their entireties). However, through local targeting and sustained release in the lower GI tract, the ButM formulations of the present invention are expected to achieve greater therapeutic potential in food allergies and more broadly.

[0096] This approach is not antigen-specific and can therefore be easily extended to other food allergens, such as nuts, milk, eggs, soy, and shellfish. Furthermore, the platform can also be easily adapted to deliver other SCFAs or other microbiota-derived metabolites, either in single forms or in combination, providing a more controlled and accessible method for achieving potential therapeutic efficacy.

[0097] In a first aspect, provided herein are copolymers (and micelles thereof) comprising methacrylic acid (MAA) monomers (or blocks thereof) and prodrug-containing monomers (e.g., having SCFA side chains).

[0098] In a second aspect, provided herein is a composition comprising a first copolymer assembly (e.g., a first micelle) comprising a first copolymer comprising a methacrylic acid (MAA) monomer (or a block thereof) and a prodrug-containing monomer (e.g., having an SCFA side chain), and a second copolymer assembly (e.g., a second micelle) comprising a second copolymer comprising an N-(2-hydroxyethyl)methacrylamide (HPMA) monomer (or a block thereof) and a prodrug-containing monomer (e.g., having an SCFA side chain).

[0099] In a third aspect, provided herein are pharmaceutical or nutraceutical compositions comprising the copolymers herein and noncovalent assemblies (e.g., micelles) thereof (e.g., (1) MAA / prodrug copolymers and micelles, (2) MAA / prodrug copolymer micelles and HPMA / prodrug copolymer micelles, etc.), as well as methods of administering such pharmaceutical or nutraceutical compositions, for example, for the treatment or prevention of inflammatory, autoimmune, allergic, metabolic, and central nervous system diseases, or for modulation of microbiota levels.

[0100] In a fourth aspect, provided herein are methods for administering compositions described herein to detect / monitor and modify or regulate levels of metabolic and / or microbiota biomarkers in a subject (e.g., for the treatment or prevention of inflammatory, autoimmune, allergic, metabolic, central nervous system disorders, etc.).

[0101] In some embodiments, provided herein are (e.g., block or random) copolymers comprising methacrylic acid (MAA) monomers (or blocks thereof) and prodrug monomers (e.g., comprising SCFA side chains). In some embodiments, provided are methods for assembling these copolymers into nanoparticles, micelles, or other delivery systems. In some embodiments, provided are methods for administering the copolymers, as well as delivery systems comprising such copolymers, for the treatment or prevention of various diseases and disorders. In particular, the polymers are functionalized with covalent bonds that are cleaved (e.g., by hydrolysis or enzymatic activity) to deliver pharmaceutically suitable small molecule moieties (e.g., SCFAs) relevant to the treatment of human diseases.

[0102] In some embodiments, a (e.g., block or random) copolymer comprising methacrylic acid (MAA) monomers (or blocks thereof) and prodrug monomers (e.g., comprising SCFA side chains), or aggregates thereof (e.g., micelles thereof) (e.g., as part of a composition or system thereof) is provided, as is a (e.g., block or random) copolymer comprising N-(2-hydroxyethyl)methacrylamide (HPMA) monomers (or blocks thereof) and prodrug monomers (e.g., comprising SCFA side chains), or aggregates thereof (e.g., micelles thereof).

[0103] In some embodiments, the copolymers herein are obtained using reversible addition-fragmentation chain transfer ("RAFT") polymerization of suitable monomers with an initiator.

[0104] In some embodiments, the free terminus of the polymer may be one of many chemical groups, including, but not limited to, hydroxyl, methoxy, benzyl, cyano, thiol, amine, maleimide, halogen, polymer chain transfer agent, protecting group, drug, biomolecule, or tissue-targeting moiety. Some or all of the polymers expose a pharmaceutically suitable small molecule covalently attached to the hydroxyethyl functional group. Preferred embodiments of the pharmaceutically suitable small molecule are short- and medium-chain fatty acids ("SCFAs") and their derivatives containing up to 12 carbon atoms in the chain, e.g., between 3 and 10 carbon atoms in the chain. The chain may be linear or branched. Exemplary SCFAs include, but are not limited to, acetate, propionate, isopropionate, butyrate, isobutyrate, and other SCFAs described herein, and their derivatives. The free SCFA terminus may be one of many chemical groups, including, but not limited to, methyl, hydroxyl, methoxy, thiol, amine, N-alkylamine, etc.

[0105] In some embodiments, the MAA or HPMA copolymer is a copolymer (e.g., a random copolymer) of MAA or HPMA monomers and N-hydroxyethyl methacrylate monomers. The free MAA or HPMA termini may be one of many chemical groups, including, but not limited to, hydroxyl, cyano, benzyl, methoxy, thiol, amine, maleimide, halogen, polymer chain transfer agent, protecting group, drug, biomolecule, or tissue-targeting moiety. Some or all of the N-hydroxyethyl methacrylate monomers expose a pharmaceutically suitable small molecule covalently attached to the hydroxyethyl functional group. Preferred embodiments of the pharmaceutically suitable small molecule are short- and medium-chain fatty acids ("SCFAs") and their derivatives, for example, containing between 3 and 12 carbon atoms in the chain (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or any range therebetween). The chain may be linear or branched. Exemplary SCFAs include, but are not limited to, acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, and other SCFAs described herein, and derivatives thereof. The free SCFA terminus may be one of many chemical groups, including, but not limited to, methyl, hydroxyl, methoxy, thiol, amine, N-alkylamine, etc.

[0106] The blocks may vary in molecular weight, and therefore size, to alter the ratio of inert, non-functionalized, pharmaceutically inactive material to active, functionalized, pharmaceutically active material. Some embodiments are linear MAA or HPMA block copolymers having relative block sizes between 0.25 and 3.5 (e.g., 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, and ranges therebetween (e.g., 0.7-1.8)). Other embodiments are linear MAA block copolymers with relative block sizes between 0.25 and 3.5 (e.g., 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, and ranges therebetween, e.g., 0.7 to 1.8). A "relative block size" of 0.25 to 3.5 means that for every mole of MAA or HPMA by weight, there are 0.25 to 3.5 moles of N-oxyethylmethacrylamide block (or SCFA-functionalized derivative). In some embodiments, the block copolymers described herein, when dispersed (e.g., in a liquid), form nanoparticles or micelles of 10 to 1000 nm (e.g., 10, 20, 50, 100, 200, 500, 1000 nm, or any range therebetween (e.g., 50 to 500 nm). The nanoparticles or micelles thus formed can then be isolated as a solid (e.g., powder, such as by lyophilization) with or without a stabilizer (e.g., a surfactant).

[0107] The MAA or HPMA blocks may be present in molecular weights between 3000 and 50,000 Da (e.g., 3000, 4000, 5000 Da, 6000 Da, 7000 Da, 8000 Da, 9000 Da, 10000 Da, 11000 Da, 12000 Da, 13000 Da, 14000 Da, 15000 Da, 20000 Da, 25000 Da, 30000 Da, 35000 Da, 40000 Da, 45000 Da, 50000 Da, or ranges therebetween (e.g., 9000-14000 Da, 14000-30000)).

[0108] In some embodiments, described herein is a monomer comprising (i) methacrylic acid (MAA) and (ii) a monomer of formula (I): [ka] wherein X is O, NH, or S, and L is a linker selected from an alkyl chain, a heteroalkyl chain, a substituted alkyl chain, or a substituted heteroalkyl chain, wherein the copolymer has one or more exposed short chain fatty acid (SCFA) moieties.

[0109] In some embodiments, described herein is a monomer comprising (i) methacrylic acid (MAA) and (ii) a monomer of formula (II): [ka] Compositions are provided that include copolymers with monomers of formula (II), wherein X is O, NH, or S; L is a linker selected from an alkyl chain, a heteroalkyl chain, a substituted alkyl chain, or a substituted heteroalkyl chain; and SCFA is a short chain fatty acid.

[0110] In some embodiments, L in Formula (I) or Formula (II) is (CH) nwherein n is 1 to 16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a range therebetween). In some embodiments, L is (CH2). n O(CO)-benzene. In some embodiments, the SCFA is covalently bound to the monomer of formula (I). In some embodiments, the SCFA bound to the monomer of formula (I) comprises a monomer of formula (II). [ka] In some embodiments, the SCFA attached to the monomer of formula (I) or the monomer of formula (II) is of formula (III): [ka] In some embodiments, the SCFA is selected from the group consisting of acetic acid, propionic acid, isopropionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid, caprylic acid, capric acid, lauric acid, branched chain forms thereof, and derivatives thereof. In some embodiments, the SCFA is butyric acid.

[0111] In some embodiments, the copolymer is a block copolymer comprising an MAA block and a block of formula (I) or (II). In some embodiments, the block copolymer is a block copolymer comprising a block of formula (IV): [ka] wherein M h contains MAA, and M F2 is the side chain of the monomer of formula (II), [ka] a is 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or a range therebetween), and b is 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or a range therebetween).

[0112] In some embodiments, the copolymer is a random copolymer. In some embodiments, the random copolymer comprises formula (V): [ka] wherein each Y independently represents a side chain of a polymer formed from formula (II): [ka] and The side chain of MAA, [ka] In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0113] In some embodiments, the monomer of Formula (II) comprises N-butanoyloxyalkyl methacrylamide. In some embodiments, the N-butanoyloxyalkyl methacrylamide monomer is 2-butanoyloxyethyl methacrylamide. In some embodiments, the copolymer is a block copolymer and comprises Formula (VI): [ka] wherein a and b are independently 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or ranges therebetween). In some embodiments, the copolymer is a random copolymer and comprises formula (V): [ka] wherein each Y independently represents (i) the side chain of MAA; [ka] and (ii) side chains of poly(2-butanoyloxyethyl methacrylamide). In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0114] In some embodiments, the monomer of Formula (II) comprises N-butanoyloxyalkyl methacrylate. In some embodiments, the N-butanoyloxyalkyl methacrylate monomer is 2-butanoyloxyethyl methacrylate. In some embodiments, the copolymer is a block copolymer and comprises Formula (VII): [ka] wherein a and b are independently 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or ranges therebetween). In some embodiments, the copolymer is a random copolymer and comprises formula (V): [ka] wherein each Y independently represents (i) the side chain of MAA; [ka] and (ii) the side chains of poly(2-butanoyloxyethyl methacrylate). In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0115] In some embodiments, the monomer of formula (II) comprises an N-(4-butanoyloxybenzoyloxy)alkyl methacrylate. In some embodiments, the N-(4-butanoyloxybenzoyloxy)alkyl methacrylate monomer is 2-(4-butanoyloxybenzoyloxy)ethyl methacrylate. In some embodiments, the copolymer is a block copolymer and comprises formula (VIII): [ka] wherein a and b are independently 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or ranges therebetween). In some embodiments, the copolymer is a random copolymer and comprises formula (V): [ka] wherein each Y independently represents (i) the side chain of MAA; [ka] and (ii) side chains of poly(2-(4-butanoyloxybenzoyloxy)ethyl methacrylate), [ka] In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0116] In some embodiments, the monomer of formula (II) comprises N-(4-butanoyloxybenzoyloxy)alkyl methacrylamide. In some embodiments, the N-(4-butanoyloxybenzoyloxy)alkyl methacrylamide monomer is 2-(4-butanoyloxybenzoyloxy)ethyl methacrylamide. In some embodiments, the copolymer is a block copolymer and comprises formula (IX): [ka] wherein a and b are independently 1 to 1000 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 133, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or ranges therebetween). In some embodiments, the copolymer is a random copolymer and comprises formula (V): [ka] wherein each Y independently represents (i) the side chain of MAA; [ka] and (ii) side chains of poly(2-(4-butanoyloxybenzoyloxy)ethyl methacrylamide); [ka] In some embodiments, there are 2, 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or any range therebetween, repeated exposed groups Y.

[0117] In certain embodiments, the copolymer compositions herein are administered in the form of a pharmaceutical composition, a dietary supplement, or a food or drink. When the compositions herein are used as a food or drink, the food or drink may be, for example, a health food, a functional food, a food for specific health uses, a dietary supplement, or a patient food. The compositions may be administered once or more than once. When administered multiple times, the compositions may be administered periodically (e.g., twice a day, once a day, once every two days, once a week, once a month, once a year), as needed, or irregularly. The administration frequency of the compositions can be empirically determined by those skilled in the art.

[0118] The release of the pharmaceutically active small molecule (e.g., SCFA) is necessarily an important aspect of the copolymer's performance for material processing or downstream biological applications. In some embodiments, the pharmaceutically active small molecule can be cleaved from the polymer backbone under appropriate biological conditions, including hydrolysis (e.g., at a specific pH) and enzymatic activity (e.g., esterases). In this regard, the copolymer may be referred to as a prodrug. In various embodiments, the pharmaceutical composition comprises about 10-80% by weight (e.g., 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% by weight, or any range therebetween) of the pharmaceutically active small molecule, e.g., SCFA, or a derivative thereof. Those skilled in the art of clinical pharmacology can readily determine dosage amounts using routine experimentation.

[0119] The release of such pharmaceutically active small molecules necessarily has therapeutic effects that mimic those of SCFAs, including targeting the intestine and the barrier function of the intestinal mucus layer, and can treat all diseases in which SCFAs have been implicated in therapeutic benefits, such as increasing the thickness or barrier function of the mucus layer. In some embodiments, treatable human diseases include, but are not limited to, rheumatoid arthritis, celiac disease and other autoimmune diseases, all types of food allergies, eosinophilic esophagitis, allergic rhinitis, allergic asthma, pet allergies, drug allergies, and other allergic and atopic diseases, inflammatory bowel disease, ulcerative colitis, Crohn's disease, and additional inflammatory diseases, infectious diseases, metabolic disorders, multiple sclerosis, Alzheimer's disease, Parkinson's disease, dementia, and other diseases of the central nervous system, thalassemia and other blood disorders, colon cancer, diarrhea and related disorders affecting intestinal motility, type 1 diabetes, and autism spectrum disorders, among others. This list is not exhaustive and those skilled in the art will readily be able to treat additional indications for which SCFAs have been shown to have therapeutic benefits.

[0120] Pharmaceutical formulations can be prepared from the compositions of the present invention using methods known to those skilled in the art. The formulations are prepared using pharmaceutically acceptable "carriers" composed of materials deemed safe and effective without causing undesirable biological side effects or interactions. Suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerin, ethanol, and combinations thereof. The compositions can be adapted to the mode of administration and may be in the form of, for example, a pill, tablet, capsule, spray, powder, or liquid. In some embodiments, the pharmaceutical compositions contain one or more pharmaceutically acceptable excipients appropriate for the selected route and mode of administration, such as coating agents, fillers, binders, lubricants, disintegrants, stabilizers, or surfactants. These compositions can be administered by any parenteral route, including, but not limited to, intravenous, intraarterial, intramuscular, subcutaneous, intradermal, intraperitoneal, and intrathecal, as well as topically, orally, and by mucosal delivery routes, such as intranasal, inhalation, rectal, vaginal, buccal, and sublingual. In some embodiments, pharmaceutical compositions of the invention are prepared for administration to a vertebrate (e.g., mammalian) subject in the form of a solution, including a sterile, non-pyrogenic solution for injection, an emulsion, a powder, an aerosol, a tablet, a capsule, an enteric-coated tablet, or a suppository.

[0121] In some embodiments, compositions and methods are provided for establishing (e.g., re-establishing (e.g., after, during, or after medical treatment (e.g., chemotherapy, antibiotics, etc.)) a healthy gut microbiota in a subject. In some embodiments, compositions and methods are provided for treating diseases or disorders (e.g., autoimmune diseases (e.g., rheumatoid arthritis, celiac disease), allergic and atopic diseases (e.g., all types of food allergies, eosinophilic esophagitis, allergic rhinitis, allergic asthma, pet allergies, drug allergies), inflammatory diseases (e.g., inflammatory bowel disease, ulcerative colitis, Crohn's disease), etc.) through the establishment of a healthy gut microbiota. In some embodiments, administration of a composition herein promotes the growth of commensal gut bacteria (e.g., a bacterial species of the family Lachnospiraceae (e.g., the bacteria is of Clostridium cluster XIVa, IV, and / or XVIII). In some embodiments, administration of a composition herein inhibits the growth of pathogenic bacteria. In some embodiments, the methods herein include assessing the level of gut bacteria (e.g., commensal bacteria, pathogenic bacteria, etc.) in the subject (e.g., in the subject's stool). In some embodiments, the level of gut bacteria is assessed before treatment with a composition herein and / or after treatment with a composition herein.

[0122] In some embodiments, compositions and methods are provided for establishing healthy levels of gut metabolites in a subject (e.g., re-establishing (e.g., after, during, or after medical treatment (e.g., chemotherapy, antibiotics, etc.)). In some embodiments, compositions and methods are provided for treating a disease or disorder (e.g., autoimmune diseases (e.g., rheumatoid arthritis, celiac disease), allergic and atopic diseases (e.g., all types of food allergies, eosinophilic esophagitis, allergic rhinitis, allergic asthma, pet allergies, drug allergies), inflammatory diseases (e.g., inflammatory bowel disease, ulcerative colitis, Crohn's disease), etc.) through establishing healthy levels of gut metabolites. In some embodiments, administration of a composition herein provides beneficial metabolites (e.g., SCFAs (e.g., butyrate)) and promotes assimilation of beneficial metabolites in the subject. In some embodiments, a method herein includes assessing the level of a metabolite (e.g., SCFAs (e.g., butyrate)) in the subject (e.g., the subject's stool). In some embodiments, the levels of the intestinal metabolites are assessed before treatment with a composition herein and / or after treatment with a composition herein.

[0123] experiment Example 1 pMAA-b-pBMA Block copolymers capable of forming water-suspendable micelles with a high butyrate content within the core for delivery of SFCA (e.g., butyrate ester) to the gastrointestinal (GI) tract have been previously described (U.S. Publication No. 2020 / 0048390, incorporated by reference in its entirety). Provided herein is an exemplary copolymer, pMAA-b-pBMA, which has an anionic block made of hydrophilic methacrylic acid (MAA) and spontaneously forms negatively charged micelles (Neg-ButM) in alkaline aqueous solutions. The negative surface charge affects distribution and absorption upon intragastric administration. Notably, Neg-ButM exhibited slower release kinetics in simulated gastric fluid, longer retention time in the GI tract, and greater butyrate ester release in the mouse cecum compared to neutrally charged NtL-ButM.

[0124] During development of embodiments herein, experiments were conducted to investigate lymph node targeting of negatively charged Neg-ButM when injected subcutaneously (SC). Neg-ButM showed excellent accumulation and long-term retention in the draining LNs after SC administration, resulting in substantial induction of regulatory T cells (Tregs). This effect may be useful in several inflammatory and immunological medical diseases.

[0125] Synthesis of pMAA-b-BMA Synthesis of N-(2-hydroxyethyl)methacrylamide (2) To synthesize N-(2-hydroxyethyl)methacrylamide (HEMA, 2), ethanolamine (3.70 mL, 61.4 mmol, 2.0 equiv.), triethylamine (4.72 mL, 33.8 mmol, 1.1 equiv.), and 50 mL of DCM were added to a 250 mL flask. After cooling the system in an ice bath, methacryloyl chloride (1, 3.00 mL, 30.7 mmol, 1.0 equiv.) was added dropwise under nitrogen protection. The reaction mixture was allowed to warm to room temperature and react overnight. The reaction mixture was then concentrated by rotary evaporation and purified on a silica column using DCM / MeOH (0% to 5% v / v MeOH ratio). The product was obtained as a colorless oil (3.42 g, 86.3%). MS (ESI). m / z calculated for C6H11NO2, [M+H]+: 129.08, found: 129.0. 1H-NMR (500 MHz, CDCl3) δ 1.93 (s, 3H), 3.43 (m, 2H), 3.71 (m, 2H), 5.32 (s, 1H), 5.70 (s, 1H), 6.44 (br s, 1H).

[0126] Synthesis of N-(2-butanoyloxyethyl)methacrylamide (3) To synthesize N-(2-butanoyloxyethyl)methacrylamide (BMA, 3), N-(2-hydroxyethyl)methacrylamide (3.30 mL, 25.6 mmol, 1.0 equiv.), triethylamine (7.15 mL, 51.2 mmol, 2.0 equiv.), and 50 mL of DCM were added to a 250 mL flask. After cooling the reaction in an ice bath, butyric anhydride (5.00 mL, 30.7 mmol, 1.2 equiv.) was added dropwise under nitrogen protection. The reaction mixture was allowed to react overnight. The reaction mixture was filtered and washed with NHCl solution, NaHCO solution, and water. After drying over anhydrous MgSO, the organic layer was concentrated by rotary evaporation and purified on a silica column using DCM / MeOH (0% to 5% v / v MeOH ratio). The product was obtained as a pale yellow oil (4.56 g, 89.6%). MS(ESI).C10H17NO3, m / z calculated with [M+H]+: 199.12, actual value: 199.1. 1H-NMR (500 MHz, CDCl3) δ 0.95 (t, 3H), 1.66 (m, 2H), 1.97 (s, 3H), 2.32 (t, 2H), 3.59 (dt, 2H), 4.23 (t, 2H), 5.35 (s, 1H), 5.71 (s, 1H), 6.19 (br s, 1H)

[0127] Synthesis of pMAA(7) and pMAA-b-pBMA(8) pMAA (7) was prepared using 2-cyano-2-propyl dithiobenzoate as the RAFT chain transfer agent and 2,2'-azobis(2-methylpropionitrile) (AIBN) as the initiator. Briefly, in a 50 mL Schlenk tube, methacrylic acid (MAA) (4.0 mL, 47.2 mmol, 1.0 equiv.), 2-cyano-2-propyl dithiobenzoate (104.4 mg, 0.472 mmol, 1 / 100 equiv.), and AIBN (19.4 mg, 0.118 mmol, 1 / 400 equiv.) were dissolved in 20 mL of MeOH. The reaction mixture was subjected to four freeze-pump-thaw cycles. Polymerization was carried out at 70 °C for 24 h. The polymer was precipitated in hexane and dried overnight in a vacuum oven. The product was obtained as a pale pink solid (4.0 g, 100%). 1H-NMR (500 MHz, DMSO-d6) δ 0.8-1.2 (m, 3H, main chain CH3), 1.5-1.8 (m, 2H, backbone CH2), 7.4-7.8 (three peaks, 5H, aromatic H), 12.3 (m, 1H, CO-OH)

[0128] The block copolymer pMAA-b-pBMA (8) was prepared using (7) pMAA as the macro-RAFT chain transfer agent and (3) N-(2-butanoyloxyethyl)methacrylamide (BMA) as the monomer in the second RAFT polymerization. Briefly, in a 25 mL Schlenk tube, pMAA (0.50 g, 0.058 mmol, 1.0 equiv.), N-(2-butanoyloxyethyl)methacrylamide (1.47 g, 7.38 mmol, 127 equiv.), and AIBN (2.4 mg, 0.015 mmol, 0.25 equiv.) were dissolved in 10 mL of MeOH. The reaction mixture was subjected to four freeze-pump-thaw cycles. The polymerization was carried out at 70 °C for 24 h. The polymer was precipitated in hexane and dried overnight in a vacuum oven. The product was obtained as a pale pink solid (1.5 g, 70%). 1H-NMR (500 MHz, DMSO-d6) δ 0.8-1.1 (m, 6H, CH2-CH3 (BMA), and backbone CH3), 1.5-1.7 (m, 4H, CH2-CH2 (BMA) and backbone CH2), 2.3 (m, 2H, CO-CH2 (BMA)), 3.2 (m, 2H, NH-CH2 (BMA)), 4.0 (m, 2H, O-CH2 (BMA)), 7.4 (m, 1H, NH), 12.3 (m, 1H, CO-OH)

[0129] [ka]

[0130] [ka]

[0131] [ka]

[0132] Manufacturing Neg-ButM Neg-ButM micelles were prepared by base titration (see References A8 and A9; incorporated by reference in their entirety). 60 mg of pMAA-b-pBMA polymer was added to 8 mL of 1x PBS under vigorous stirring. Sodium hydroxide solution equivalent to the amount of methacrylic acid was added to the polymer solution in three portions over a 2-hour period. After the base solution was added, the polymer solution was stirred overnight at room temperature. 1x PBS was then added to reach the target volume, and the solution was filtered through a 0.22 μm filter. The pH of the solution was checked to confirm neutrality. The size of the micelles was measured by dynamic light scattering (DLS).

[0133] pMAA-b-pBMA cannot be formulated into micelles by this method due to the formation of intramolecular hydrogen bonds between pMAA chains (Reference A10; incorporated by reference in its entirety). However, titration of a strong base, here NaOH, into a mixture of pMAA-b-pBMA polymers can disrupt these bonds and convert methacrylic acid to ionized methacrylate esters (References A8, A9, and A11; incorporated by reference in their entireties). Upon base titration, pMAA-b-pBMA polymers can self-assemble into negatively charged micelles (Neg-ButM) with diameters of 39.9 ± 1.6 nm, as measured by dynamic light scattering (DLS). Their low polydispersity index of less than 0.1 indicated the monodispersity of these micelles (Figure 5). Neg-ButM has a zeta-potential of -31.5 ± 2.3 mV, due to the ionization of methacrylic acid. It has been reported that negatively charged nanoparticles can remain in the GI tract for longer periods due to their stronger adhesive effect on the intestinal mucosa (References A10, A12-A13; incorporated by reference in their entirety). Additionally, cryo-electron microscopy (cryoEM) revealed detailed structures of micelles, especially the core structure made of pBMA. The micelles were more condensed with higher contrast. CryoEM images showed that the core diameter of NtL-ButM was 30 nm, while Neg-ButM had a smaller core diameter of 15 nm (Figure 5C, D). To obtain the critical micelle concentration (CMC) of NtL-ButM and Neg-ButM, which indicates the possibility of micelle formation and dissociation in aqueous solution, pyrene was added during formulation, and the CMC was calculated by plotting the fluorescence intensity ratio between the first and third vibronic bands of pyrene (References A14-A15; incorporated by reference in their entirety). The results showed that Neg-ButM had a higher CMC of 14.0 ± 3.5 μM compared to that of NtL-ButM, which was 0.8 ± 0.4 μM (Figure 5E). The higher CMC indicated that Neg-ButM micelles dissociated more easily in solution, likely because the surface charge destabilized the micellar structure compared to the neutral micellar NtL-ButM.In addition, we performed small-angle X-ray scattering (SAXS) analysis on both micelles to obtain the aggregation numbers, which were 119 for NtL-ButM and 92 for Neg-ButM (Figure 5E).

[0134] In vitro release kinetics Simulated gastric and intestinal fluids were as previously described (see References A16-A17; incorporated by reference in their entirety). For ex vivo hydrolysis studies, NtL-ButM or Neg-ButM was added to simulated gastric or intestinal fluid at a final concentration of 2 mg / mL at 37°C. At predetermined time points, 20 μL of the solution was transferred to 500 μL of water:acetonitrile (1:1 v / v). The samples were centrifuged at 13,000 × g for 15 min using an Amicon Ultra (Merck, 3 kDa molecular weight cutoff) to remove polymers. The filtrate was stored at -80°C before derivatization.

[0135] Samples were prepared and derivatized as described in the literature (see References A18-A19; incorporated by reference in their entirety). A 3-nitrophenylhydrazine (NPH) stock solution was prepared at 0.02 M in water:acetonitrile (1:1 v / v). A 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) stock solution was prepared at 0.25 M in water:acetonitrile (1:1 v / v). 4-Methylvaleric acid was added as an internal standard. Samples were mixed with the NPH and EDC stock solutions in a 1:1:1 volume ratio. The mixture was heated by heating the block at 60°C for 30 minutes. Samples were transferred to HPLC vials and stored at 4°C before analysis.

[0136] LC conditions: The instrument used for the quantification of butyrate esters was an Agilent 1290U HPLC. Column: Thermo Scientific C18 4.6 x 50 mm, 1.8 μm particle size, room temperature. Mobile phase A: water with 0.1% v / v formic acid. Mobile phase B: acetonitrile with 0.1% v / v formic acid. Injection volume: 5.0 μL. Flow rate: 0.5 mL / min. Solvent gradient: 15% mobile phase B at 0.0 min, 100% mobile phase B at 3.5 min, 100% mobile phase B at 6.0 min, 15% mobile phase B at 6.5 min.

[0137] Liquid chromatography with tandem mass spectrometry (LC-MS / MS) method: The instrument used to detect butyrate esters was an Agilent 6460 Triple Quad MS-MS. Both derivatized butyrate ester-NPH and 4-methylvalerate-NPH were detected in negative mode. MS conditions were optimized for pure butyrate ester-NPH or 4-methylvalerate-NPH at 1 mM. The fragment voltage was 135 V, and the collision energy was set at 18 V. The multiple reaction monitoring (MRM) from 222 to 137 was assigned to butyrate ester, and the MRM from 250 to 137 was assigned to 4-methylvaleric acid as an internal standard. The concentration of butyrate ester was quantified using the ratio between the MRM of butyrate ester and 4-methylvaleric acid.

[0138] In simulated gastric fluid, both Neg-ButM and NtL-ButM showed a slight release of butyrate esters within a few hours and a sustained release over 3 weeks, although Neg-ButM had a slower release rate than NtL-ButM (Figure 6A). The anionic surface of Neg-ButM in an acidic environment likely contributes to the resistance of the BMA core to hydrolysis. In contrast, in simulated intestinal fluid, both micelles released most of the butyrate esters within minutes in the presence of high concentrations of the esterase pancreatin (Figure 6B).

[0139] In vivo biodistribution and pharmacokinetics To investigate how butyrate esters are delivered from these micelles upon oral administration, we first studied the biodistribution of fluorescently labeled NtL-ButM and Neg-ButM in mice via an in vivo imaging system (IVIS) (Figure 7). IVIS results verified that the polymeric micelles were retained in the mouse GI tract for more than 6 h after gavage. Neutral micellar NtL-ButM passed through the stomach and small intestine within 2 h and accumulated in the cecum. However, negatively charged Neg-ButM first accumulated in the stomach and then gradually moved through the small intestine to the cecum. Overall, Neg-ButM had a longer retention time in the stomach and small intestine. Both micelles were excreted from the GI tract within 24 h after administration. In addition, fluorescent signals from other major organs and plasma were measured by IVIS, and plasma butyrate ester concentrations were measured by LC-MS / MS. All signals were below the detection limit from both methods, suggesting that there was minimal absorption of these butyrate micelles from the intestine into the circulation, consistent with our desire to deliver butyrate to the intestine and avoid the complications of systemic absorption of polymers or micelles.

[0140] We also measured butyrate levels in the GI tract of mice affected by polymeric micelles. Using both LC-UV and LC-MS / MS methods, we measured butyrate concentrations in the fecal contents of the ileum, cecum, or colon of mice orally administered either NtL-ButM or Neg-ButM (see References A18–A19; incorporated by reference in their entirety). We primarily used the LC-MS / MS method to measure butyrate concentrations in the ileum, because baseline concentrations in the ileum were too low for the UV detector. Results showed that NtL-ButM dramatically increased butyrate concentrations in the ileum for up to 2 h after gavage, but this was short-lived; butyrate concentrations did not increase in either the cecum or colon (Figure 8). Interestingly, Neg-ButM increased butyrate concentrations in the cecum threefold, starting 4 h after gavage and sustained for at least another 8 h. This increase, however, did not increase butyrate concentrations in the ileum or colon. Due to the different butyrate release behavior in vivo from the two butyrate micelles, the combined dose of NtL-ButM and Neg-ButM can cover most compartments of the GI tract and can be sustained for a longer period of time when applied in animal disease models.

[0141] Neg-ButM accumulated in the draining LN after subcutaneous (SC) injection Lymphatic vessels exhibit wider interendothelial junctions than vascular capillaries, allowing larger carriers (10–100 nm) to enter the interstitium more efficiently (Reference A20; incorporated herein by reference in its entirety). In addition, neutral or positively charged vehicles are more likely to be trapped in the negatively charged interstitial extracellular matrix (Reference A21; incorporated herein by reference in its entirety). Herein, we demonstrate the use of Neg-ButM as a novel platform for targeting butyrate esters to LNs via SC administration. To track the in vivo biodistribution of micelles, we fluorescently labeled the polymer and administered either NtL-ButM, Neg-ButM, or equivalent free dye subcutaneously into the abdomen of specific pathogen-free (SPF) C3H / HeJ mice. At various time points after injection, blood and tissues were collected, and the fluorescent signal was quantified using an in vivo imaging system (IVIS). We also digested LNs and spleens into single-cell suspensions and analyzed the cellular biodistribution using flow cytometry. We observed that Neg-ButM accumulated and was retained in the draining inguinal LNs for a very long time (more than 35 days), whereas NtL-ButM was not (Figures 9A and 9B). At the cellular level, both micelles were primarily taken up by macrophages in the LNs, but Neg-ButM was taken up by more cells than NtL-ButM (Figure 9C).

[0142] Neg-ButM inhibited LPS-induced activation of APCs in the dLN. To evaluate the ability of Neg-ButM to target lymph nodes and inhibit APC activation, we tested it in a mouse model of subcutaneous lipopolysaccharide (LPS) stimulation and assessed activation markers, including CD40 and CD86, on primary APCs in the draining LN. Mice were injected subcutaneously in the abdomen with either PBS, sodium butyrate (NaBut), NtL-ButM, or Neg-ButM. On day 6, mice were challenged with LPS at the same injection site and sacrificed the following day for cellular analysis.

[0143] Neg-ButM treatment significantly inhibited the overexpression of CD40 and CD86 on subcapsular macrophages and CD169-CD11b+F4 / 80+ macrophages in the draining LN upon LPS stimulation (Figure 10). These macrophages were also the major uptakers of Neg-ButM from previous cell biodistribution studies. Neg-ButM also reduced CD86 expression on CD11b+ dendritic cells. In contrast, neither NtL-ButM nor sodium butyrate significantly suppressed APC activation.

[0144] Neg-ButM induced suppressive Tregs in the draining LNs. Microbiota-derived short-chain fatty acids (SCFAs) have been shown to promote extrathymic differentiation of Tregs (see References A5-A6; incorporated by reference in their entirety). Due to the ability of acetates to induce gut-localized Tregs, butyrates in particular have long been considered promising therapeutic candidates (see References A5-A6; incorporated by reference in their entirety).

[0145] Peripherally derived Tregs are most efficiently induced in lymph nodes (LNs), but current therapies do not efficiently target LNs. Herein, we demonstrated Treg induction in draining LNs through SC administration of Neg-ButM. To investigate the potential for Treg induction in vivo, we administered PBS, Neg-ButM, or Neg-ButM to SPF C57BL / 6 Foxp3 mice by weekly SC injection for 3 weeks. GFP+Mice were administered Neg-ButM. One week after the final administration, the mice were sacrificed, and cells were isolated from the relevant LNs and spleens to analyze the Treg population by flow cytometry (Figure 11A). We also measured butyrate concentrations in the liver, spleen, serum, and colon contents via LC-MS / MS. We demonstrated that antibiotic-treated mice had lower Treg frequencies in both the LNs and spleens than SPF mice. However, Neg-ButM treatment significantly increased and restored the Treg population in the draining inguinal LNs compared with PBS- or NtL-ButM-treated mice. In SPF mice, Neg-ButM further increased the Treg population in the draining LN (Fig. 11B), and a substantial increase in RORt+ Tregs, a subset of Tregs induced by gut microbes or SCFAs that suppresses Th2 responses, was also observed in the draining LN (Fig. 11C) (Reference A22; incorporated by reference in its entirety). Micelles also released butyrate esters in the liver and spleen (Fig. 11D), suggesting their potential to enter the systemic circulation.

[0146] Example 2 Combined pHPMA-b-pBMA and pMAA-b-pBMA micelles I. Materials and Methods Materials for polymer synthesis N-(2-hydroxyethyl)methacrylamide (HPMA) monomer was obtained from Sigma-Aldrich or Polysciences, Inc. Solvents, including dichloromethane, methanol, hexane, and ethanol, were ACS reagent grade and were obtained from Fisher Scientific. All other chemicals were obtained from Sigma-Aldrich.

[0147] Synthesis of N-(2-hydroxyethyl)methacrylamide (2) To synthesize N-(2-hydroxyethyl)methacrylamide (HEMA, 2), ethanolamine (3.70 mL, 61.4 mmol, 2.0 equiv.), triethylamine (4.72 mL, 33.8 mmol, 1.1 equiv.), and 50 mL of DCM were added to a 250 mL flask. After cooling the system in an ice bath, methacryloyl chloride (1, 3.00 mL, 30.7 mmol, 1.0 equiv.) was added dropwise under nitrogen protection. The reaction mixture was allowed to warm to room temperature and react overnight. The reaction mixture was then concentrated by rotary evaporation and purified on a silica column using DCM / MeOH (0% to 5% v / v MeOH ratio). The product was obtained as a colorless oil (3.42 g, 86.3%). MS (ESI): CHNO, [M+H] + Calculated m / z: 129.08, observed value: 129.0. 1 H-NMR (500 MHz, CDCl3) δ 1.93 (s, 3H), 3.43 (m, 2H), 3.71 (m, 2H), 5.32 (s, 1H), 5.70 (s, 1H), 6.44 (br s, 1H) (Figure 18).

[0148] [ka]

[0149] Synthesis of N-(2-butanoyloxyethyl)methacrylamide (3) To synthesize N-(2-butanoyloxyethyl)methacrylamide (BMA, 3), N-(2-hydroxyethyl)methacrylamide (3.30 mL, 25.6 mmol, 1.0 equiv.), triethylamine (7.15 mL, 51.2 mmol, 2.0 equiv.), and 50 mL of DCM were added to a 250 mL flask. After cooling the reaction in an ice bath, butyric anhydride (5.00 mL, 30.7 mmol, 1.2 equiv.) was added dropwise under nitrogen protection. The reaction mixture was allowed to react overnight. The reaction mixture was filtered and washed with NH4Cl solution, NaHCO3 solution, and water. After drying over anhydrous MgSO4, the organic layer was concentrated by rotary evaporation and purified on a silica column using DCM / MeOH (0% to 5% v / v MeOH ratio). The product was obtained as a pale yellow oil (4.56 g, 89.6%). MS(ESI).C10H17NO3, [M+H] + Calculated m / z: 199.12, observed: 199.1. 1 H-NMR (500 MHz, CDCl3) δ 0.95 (t, 3H), 1.66 (m, 2H), 1.97 (s, 3H), 2.32 (t, 2H), 3.59 (dt, 2H), 4.23 (t, 2H), 5.35 (s, 1H), 5.71 (s, 1H), 6.19 (br s, 1H) (Fig. 19).

[0150] [ka]

[0151] Synthesis of poly(2-hydroxypropylmethacrylamide) (pHPMA, 5) pHPMA was prepared using 2-cyano-2-propyl dithiobenzoate as the RAFT chain transfer agent and 2,2'-azobis(2-methylpropionitrile) (AIBN) as the initiator. Briefly, in a 25 mL Schlenk tube, HPMA (4, 3.0 g, 20.9 mmol, 1.0 equiv.), 2-cyano-2-propyl dithiobenzoate (28.3 mg, 0.128 mmol, 1 / 164 equiv.), and AIBN (5.25 mg, 0.032 mmol, 1 / 656 equiv.) were dissolved in 10 mL of MeOH. The reaction mixture was subjected to four freeze-pump-thaw cycles. Polymerization was carried out at 70 °C for 30 h. The polymer was precipitated into a large amount of petroleum ether and dried overnight in a vacuum oven. The product was obtained as a pale pink solid (1.8 g, 60%). 1 H- NMR (500 MHz, DMSO-d6) δ 0.8-1.2 (m, 6H, CH(OH)-CH3 and main chain CH3), 1.5-1.8 (m, 2H, main chain CH2), 2.91 (m, 2H, NH-CH2), 3.68 (m, 1H, C(OH)-H), 4.70 (m, 1H, CH-OH), 7.18 (m, 1H, NH) (Figure 20).

[0152] [ka]

[0153] Synthesis of pHPMA-b-pBMA (6) The block copolymer pHPMA-b-pBMA was prepared using pHPMA (5) as a macro-RAFT chain transfer agent and N-(2-butanoyloxyethyl)methacrylamide (3) as the monomer in the second RAFT polymerization. Briefly, in a 50 mL Schlenk tube, pHPMA (1.50 g, 0.105 mmol, 1.0 equiv.), N-(2-butanoyloxyethyl)methacrylamide (4.18 g, 21.0 mmol, 200 equiv.), and AIBN (8.3 mg, 0.050 mmol, 0.50 equiv.) were dissolved in 10 mL of MeOH. The reaction mixture was subjected to four freeze-pump-thaw cycles. The polymerization was carried out at 70 °C for 20 h. The polymer was precipitated in petroleum ether and dried overnight in a vacuum oven. The resulting product was a pale pink solid (4.22 g, 74%). 1 H-NMR (500 MHz, DMSO-d6) δ 0.80-1.1 (m, 9H, CH(OH)- CH3 (HPMA), CH2-CH3 (BMA), and backbone CH3), 1.55 (m, 4H, CH2-CH2 (BMA) and backbone CH2), 2.28 (m, 2H, CO-CH2 (BMA)), 2.91 (m, 2H, NH-CH2 (HPMA)), 3.16 (m, 2H, NH-CH2 (BMA)), 3.67 (m, 1H, CH(OH)-H), 3.98 (m, 2H, O-CH2 (BMA)), 4.71 (m, 1H, CH-OH (HPMA)), 7.19 (m, 1H, NH), 7.44 (m, 1H, NH) (Figure 21).

[0154] [ka]

[0155] Synthesis of pMAA(7) and pMAA-b-pBMA(8) pMAA (7) was prepared using 2-cyano-2-propyl dithiobenzoate as the RAFT chain transfer agent and AIBN as the initiator. Briefly, in a 50 mL Schlenk tube, methacrylic acid (MAA) (4.0 mL, 47.2 mmol, 1.0 equiv.), 2-cyano-2-propyl dithiobenzoate (104.4 mg, 0.472 mmol, 1 / 100 equiv.), and AIBN (19.4 mg, 0.118 mmol, 1 / 400 equiv.) were dissolved in 20 mL of MeOH. The reaction mixture was subjected to four freeze-pump-thaw cycles. Polymerization was carried out at 70 °C for 24 h. The polymer was precipitated in hexane and dried overnight in a vacuum oven. The resulting product was a pale pink solid (4.0 g, 100%). 1 H-NMR (500 MHz, DMSO-d6) δ 0.8-1.2 (m, 3H, main-chain CH3), 1.5-1.8 (m, 2H, main-chain CH2), 7.4-7.8 (three peaks, 5H, aromatic H), 12.3 (m, 1H, CO-OH) (Figure 22).

[0156] The block copolymer pMAA-b-pBMA (8) was prepared using (7) pMAA as the macro-RAFT chain transfer agent and (3) N-(2-butanoyloxyethyl)methacrylamide (BMA) as the monomer in the second RAFT polymerization. Briefly, in a 25 mL Schlenk tube, pMAA (0.50 g, 0.058 mmol, 1.0 equiv.), N-(2-butanoyloxyethyl)methacrylamide (1.47 g, 7.38 mmol, 127 equiv.), and AIBN (2.4 mg, 0.015 mmol, 0.25 equiv.) were dissolved in 10 mL of MeOH. The reaction mixture was subjected to four freeze-pump-thaw cycles. The polymerization was carried out at 70 °C for 24 h. The polymer was precipitated in hexane and dried overnight in a vacuum oven. The resulting product was a pale pink solid (1.5 g, 70%). 1H-NMR (500 MHz, DMSO-d6) δ 0.8-1.1 (m, 6H, CH2-CH3 (BMA), and main chain CH3), 1.5-1.7 (m, 4H, CH2-CH2 (BMA) and main chain CH2), 2.3 (m, 2H, CO-CH2 (BMA)), 3.2 (m, 2H, NH-CH2 (BMA)), 4.0 (m, 2H, O-CH2 (BMA)), 7.4 (m, 1H, NH), 12.3 (m, 1H, CO-OH) (Figure 23).

[0157] [ka]

[0158] Synthesis of N3-PEG4-MA (9) and azide-PEG polymers To incorporate azide groups into pHPMA-b-pBMA or pMAA-b-pBMA polymers, the monomer N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)methacrylamide (9) was synthesized and used in copolymerization with HPMA or MAA to yield hydrophilic blocks bearing azide functionality. N3-PEG4-NH2 (0.5 g, 2.14 mmol, 1.0 equiv.) and triethylamine (0.60 mL, 4.3 mmol, 2.0 equiv.) were dissolved in anhydrous DCM. The reaction mixture was cooled in an ice bath, and then methacrylic acid chloride (0.42 mL, 2.6 mmol, 1.2 equiv.) was added dropwise under nitrogen protection. The mixture was allowed to react overnight. The reaction mixture was filtered and washed with NH4Cl solution, NaHCO3 solution, and water. After drying over anhydrous MgSO4, the organic layer was concentrated by rotary evaporation and purified on a silica column using DCM / MeOH (MeOH ratio 0% to 5% v / v). The obtained product was a pale yellow oil (0.47 g, 73%). MS (ESI): C12H22N4O4, [M+H] + Calculated m / z: 287.16, observed: 287.2. 1H-NMR (500 MHz, CDCl) δ 6.35 (br, 1H), 5.70 (s, 1H), 5.32 (s, 1H), 3.55-3.67 (m, 12H), 3.52 (m, 2H), 3.38 (t, 2H), 1.97 (s, 3H) (Figure 24). The monomer N3-PEG4-MA was mixed with HPMA or MAA in a 2:98 weight:weight ratio during RAFT polymerization to give N3-pHPMA or N3-pMAA. The second block of BMA was then added to the macroinitiator to give N3-pHPMA-b-pBMA or N3-pMAA-b-pBMA, respectively. The synthetic procedure was the same as previously described.

[0159] [ka]

[0160] Synthesis of N-hexylmethacrylamide (10) and control polymers To synthesize a control polymer without butyrate esters, the monomer N-hexylmethacrylamide (11) was synthesized and used in the polymerization of the hydrophobic block. Hexanamine (5.8 mL, 46.0 mmol, 1.5 equiv.), triethylamine (4.7 mL, 33.8 mmol, 1.1 equiv.), and 50 mL of DCM were added to a 250 mL flask. After cooling the system in an ice bath, methacryloyl chloride (3.0 mL, 30.7 mmol, 1.0 equiv.) was added dropwise under nitrogen protection. The reaction mixture was allowed to warm to room temperature and react overnight. The reaction mixture was then concentrated by rotary evaporation and purified on a silica column using DCM / MeOH (0% to 5% v / v MeOH ratio). The resulting product was a colorless oil (4.6 g, 88%). MS (ESI): CHNO, [M+H] + Calculated m / z: 184.16, observed: 184.2. 1H-NMR (500 MHz, CDCl) δ 5.75 (br, 1H), 5.66 (s, 1H), 5.30 (s, 1H), 3.31 (t, 2H), 1.96 (s, 3H), 1.54 (m, 2H), 1.28–1.32 (m, 8H), 0.88 (t, 3H) (Figure 25). After the synthesis of pHPMA or pMAA, the monomer N-hexylmethacrylamide (11) was used instead of N-(2-butanoyloxyethyl)methacrylamide for the polymerization of the second block to obtain the control polymers pHPMA-b-pHMA and pMAA-b-pHMA, respectively (Figure 26). The synthetic procedure was the same as that described above.

[0161] [ka]

[0162] Formulation of polymeric micelles NtL-ButM micelles were formulated by the co-solvent evaporation method. 80 mg of pHPMA-b-pBMA polymer was dissolved in 10 mL of ethanol under stirring. After the polymer was completely dissolved, the same volume of 1x PBS was slowly added to the solution. The solution was allowed to evaporate at room temperature for at least 6 hours to remove the ethanol. After evaporation, the NtL-ButM solution was filtered through a 0.22 μm filter and stored at 4°C. The size of the micelles was measured by DLS.

[0163] Neg-ButM micelles were prepared by base titration (see references B27 and B28; their entireties are incorporated by reference). 60 mg of pMAA-b-pBMA polymer was added to 8 mL of 1x PBS under vigorous stirring. A solution of sodium hydroxide equivalent in moles to methacrylic acid was added to the polymer solution in three portions over a period of 2 hours. After the base solution was added, the polymer solution was stirred overnight at room temperature. 1x PBS was then added to reach the target volume, and the solution was filtered through a 0.22 μm filter. The pH of the solution was checked to ensure neutrality, and the size of the micelles was measured by DLS.

[0164] Dynamic light scattering (DLS) characterization of micelles DLS data were obtained using a Zetasizer Nano ZS90 (Malvern Instruments). Samples were diluted 400-fold in 1x PBS, and 700 μL was transferred to a DLS cuvette for data acquisition. DLS intensity distributions were used to determine the hydrodynamic diameter of the micelles. For zeta potential data, micelles were diluted 100-fold in 0.1x PBS (1:10 1x PBS to MilliQ water) and transferred to a disposable folded capillary zeta cell for data acquisition.

[0165] Cryo-electron microscopy imaging of micelles CryoEM images were acquired on an FEI Talos 200 kV FEG electron microscope. Polymeric nanoparticle samples were prepared in 1x PBS and diluted to 2 mg / mL with MilliQ water. 2 μL of sample solution was applied to an electron microscope grid (Agar Scientific) with a holey carbon film. The sample grid was blotted and flash vitrified in liquid ethane using an automated plunge freezing device (Vitrbot) to control humidity (100%) and temperature (20°C). Analysis was performed at -170°C using a Gatan 626 cry-specimen holder (120,000x magnification; -5 μm defocus). Digital images were recorded with an in-line Eagle CCD camera and processed with ImageJ.

[0166] Critical micelle concentration measurement The critical micelle concentrations of NtL-ButM and Neg-ButM were determined by fluorescence spectroscopy using pyrene as a hydrophobic fluorescent probe (References B30, B52; incorporated by reference in their entireties). -4 ~2.0mg mL -1 A series of polymer solutions with concentrations ranging from 1.2 x 10 -3 mg mL -1The polymer was mixed with a pyrene solution at a concentration of 1000 ppm. The emission spectrum of the sample was recorded at 20 °C on a fluorescence spectrophotometer (HORIBA Fluorolog-3) using 335 nm as the excitation wavelength. The ratio between the first (372 nm) and third (383 nm) vibronic bands of pyrene was used to plot the polymer concentration. The data were processed with Prism software and fitted using a sigmoidal model (Figure 28).

[0167] Small-angle X-ray scattering analysis of micelles SAXS samples were prepared in 1x PBS and filtered through a 0.2 μm filter. All samples were acquired at the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory. SAXS data were analyzed using Igor Pro8 software (Figure 29). To obtain the radius of gyration (Rg), data were analyzed as ln(intensity) vs. q in the low q range. 2 Rg was then calculated from the slope of the linear fit as shown in equation (1).

number

[0168] A Kratky plot of the data is shown in Iq 2 Plots of q vs. q showed the particle structure. Furthermore, the data were fitted using a polydisperse core-shell spherical model (Figures 29f and 29g). 31 From the fitting, the micelle core radius, shell thickness, and volume fraction are derived and used to calculate the micelle molecular weight and the average intermicelle distance using the following equations:

number

number

number

[0169] mouse C3H / HeN and C3H / HeJ mice were maintained in a specific pathogen-free (SPF) facility at the University of Chicago, free of Helicobacter, Pasteurella, and murine norovirus. A breeding pair of C3H / HeJ mice was originally purchased from the Jackson Laboratory. A breeding pair of C3H / HeN mice was transferred from a germ-free (GF) facility. All experimental mice were bred in-house and weaned onto a plant-based mouse diet (Purina Lab Diet 5K67®) and autoclaved water at 3 weeks of age. Mice were maintained at a room temperature of 20–24°C on a 12-hour light / dark cycle. GF C3H / He or C57BL / 6 mice were bred and housed in the Gnotobiotic Research Animal Facility (GRAF) at the University of Chicago. GF mice were maintained in Trexler-style flexible film isolation housing units (Class Biologically Clean) using Ancare polycarbonate mouse cages (catalog no. N10HT) and Teklad Pine Shavings (7088; sterilized by autoclave) at a room temperature of 20–24°C with a 12-h light / dark cycle. All experiments were performed with littermate controls. All protocols used in this study were approved by the University of Chicago Institutional Animal Care and Use Committee. FITC-dextran intestinal permeability assays in DSS-treated mice were performed by Inotiv (Boulder, CO). SPF C57BL / 6 mice were obtained from Taconic and housed in the Inotiv animal facility. The study was conducted in accordance with The Guide for the Care & Use of Laboratory Animals (8th ed.), and all policies and procedures approved by the Inotiv IACUC were adhered to.

[0170] Biodistribution studies using an in vivo imaging system (IVIS) SPF C3H / HeJ mice were used for biodistribution studies. Azide-labeled pHPMA-b-pBMA or pMAA-b-pBMA polymers were reacted with IR750-DBCO (Thermo Fisher Scientific) and purified by hexane precipitation. After formulation into micelles, fluorescently labeled NtL-ButM or Neg-ButM were administered to mice by ig gavage. After 1, 3, 6, or 24 hours, mice were euthanized, and major organs were collected. Whole-organ fluorescence was measured via an IVIS Spectrum in vivo imaging system (Perkin Elmer). Images were processed and analyzed using Living Imaging 4.5.5 (Perkin Elmer).

[0171] Butyrate derivatization and quantification using LC-UV or LC-MS / MS Simulated gastric and intestinal fluids (Fisher Scientific) were used for in vitro release analysis as previously described (see references B53-B54; incorporated by reference in their entirety). NtL-ButM or Neg-ButM was added to simulated gastric or intestinal fluid at 37°C to a final concentration of 2 mg / mL. At predetermined time points, 20 μL of the solution was transferred to 500 μL of water:acetonitrile (1:1 v / v). The sample was centrifuged at 13,000 × g for 15 min using an Amicon Ultra filter (Merck, 3 kDa molecular weight cutoff) to remove the polymer. The filtrate was stored at -80°C and then derivatized. For in vivo release studies in the mouse GI tract, NtL-ButM or Neg-ButM micellar solutions were administered i.g. to SPF C3H / HeJ mice at 0.8 mg / g body weight. Mice were euthanized 1, 2, 4, 8, 12, and 24 hours after gavage. Luminal contents from the ileum, cecum, or colon were collected in EP tubes. After adding 500 μL of 1× PBS, the mixture was vortexed, sonicated for 10 minutes, and then centrifuged at 13,000 × g for 10 minutes. The supernatant was transferred to a 0.45 μm filter and filtered. The filtered solution was stored at -80°C before derivatization.

[0172] Sample derivatization (Figure 30a): Samples were prepared and derivatized as described in the literature (Reference B32; incorporated by reference in its entirety). 3-Nitrophenylhydrazine (NPH) stock solution was prepared at 0.02 M in 1:1 v / v water:acetonitrile. EDC stock solution was prepared at 0.25 M in 1:1 v / v water:acetonitrile. 4-Methylvaleric acid was added as an internal standard. Samples were mixed with NPH and EDC stock solutions in a 1:1:1 volume ratio. The mixture was heated by heating the block at 60°C for 30 minutes. Samples were filtered through a 0.22 μm filter, transferred to HPLC vials, and stored at 4°C before analysis.

[0173] LC conditions: The instrument used for the quantification of butyrate esters was an Agilent 1290U HPLC. Column: ThermoScientific C18 4.6 x 50 mm, 1.8 μm particle size, room temperature. Mobile phase A: water with 0.1% v / v formic acid. Mobile phase B: acetonitrile with 0.1% v / v formic acid. Injection volume: 5.0 μL. Flow rate: 0.5 mL / min. Solvent gradient: 15% mobile phase B at 0.0 min, 100% mobile phase B at 3.5 min, 100% mobile phase B at 6.0 min, 15% mobile phase B at 6.5 min.

[0174] ESI-MS / MS method: The instrument used to detect butyrate ester was an Agilent 6460 Triple Quad MS-MS. Both derivatized butyrate ester-NPH and 4-methylvalerate-NPH were detected in negative mode. MS conditions were optimized for pure butyrate ester-NPH or 4-methylvalerate-NPH at 1 mM. The fragment voltage was 135 V, and the collision energy was set at 18 V. The multiple reaction monitoring (MRM) from 222 to 137 was assigned to butyrate ester (Figure 30b), and the MRM from 250 to 137 was assigned to 4-methylvaleric acid as an internal standard. The ratio between the MRM of butyrate ester and 4-methylvaleric acid was used to quantify the concentration of butyrate ester.

[0175] RNA sequencing and data analysis Starting at weaning, GF C3H / HeN mice were administered 0.8 mg / g body weight of PBS, NtL-ButM, or control polymer i.g. once daily for 1 week. Mice were then euthanized, and ileal tissue was collected and thoroughly washed. Ileal epithelial cells (IECs) were isolated from the intestinal tissue by inverting the tissue in 0.30 mM EDTA and incubated on ice for 30 minutes with agitation every 5 minutes. RNA was extracted from IECs using an RNA isolation kit (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA samples were submitted to the University of Chicago Functional Genomics Core for library preparation and sequencing on a HiSeq2500 instrument (Illumina, Inc.). 50-bp single-end (SE) reads were generated. The quality of uncorrected sequencing reads was assessed using FastQC (v0.11.5). Transcript abundance was quantified using Kallisto (v0.45.0) using Gencode gene annotations (release M18, GRCm38.p6) and summarized to the gene level using tximport (v1.12.3), Trimmed Mean of M-value (TMM) normalization, and log2 transformation. Low-expressing genes were removed (defined as counts per million mapped reads [CPM] < 3). Differentially expressed genes (DEGs) between groups of interest were detected using limma voom (v3.40.6) with precision weighting (Reference B55; incorporated by reference in its entirety). Experimental batch and sex were included as covariates for model fitting. Significance levels and fold changes were calculated using the empirical Bayes moderated t-statistical test implemented in limma. Significant DEGs were filtered by FDR-adjusted P < 0.05 and fold change ≥ 1.5 or ≤ -1.5. For visualization of a selected number of genes on an expression heatmap, a more stringent P-value cutoff (e.g., FDR-adjusted P<0.005) may be used.

[0176] Intelectin staining and microscopic imaging GF C57BL / 6 mice were administered 0.8 mg / g body weight of NtL-ButM or PBS intravenously once daily for 1 week, starting from weaning. Mice were then euthanized and perfused. Small intestinal tissue was obtained, rolled into Swiss rolls, and prepared into histological section slides. Histological section slides were fixed and stained with fluorescent anti-intelectin antibody (R&D Systems, Clone 746420) and DAPI (ProLong antifade reagent with DAPI). Slides were imaged using a Leica fluorescence microscope. Images were processed using ImageJ software, and data were plotted and analyzed using Prism software.

[0177] In vivo FITC-dextran permeability assay SPF C57BL / 6 female mice, 8–10 weeks old, were treated with 2.5% DSS in their drinking water for 7 days. Mice were given either PBS or ButM (800, 400, or 200 mg / kg) twice daily, approximately 10–12 hours apart, or 75 mg / kg CsA as a positive treatment control, administered intragastrically once daily. From day 7 onward, DSS was removed from the drinking water for the remainder of the study. On day 10, mice were fasted for 3 hours and administered 0.1 mL of FITC-dextran 4 kDa (100 mg / mL). Four hours after administration, mice were anesthetized with isoflurane, exsanguinated, and then cervically dislocated. The concentration of FITC in serum was determined by spectrofluorometry using serially diluted FITC-dextran as a standard. Serum from mice not administered FITC-dextran was used to determine background. Similar permeability assays were also performed in an antibiotic depletion model as previously described (see Reference B19, incorporated by reference in its entirety). Two-week-old SPF C57BL / 6 littermate control mice were gavaged daily with an antibiotic mixture (0.4 mg kanamycin sulfate, 0.035 mg gentamicin sulfate, 850 U colistin sulfate, 0.215 mg metronidazole, and 0.045 mg vancomycin hydrochloride in 100 μL PBS) for 7 days until weaning. At weaning, mice were then treated twice daily with either PBS or ButM (0.8 mg / g) for 7 days. After the final treatment, mice were fasted for 3 hours and administered 50 mg / kg body weight of FITC-dextran 4 kDa (50 mg / mL). 1.5 hours after administration, blood was collected via cheek bleed, and serum FITC concentrations were measured as described above.

[0178] Peanut sensitization, ButM treatment, and challenge SPF C3H / HeN mice were treated with 0.45 mg vancomycin in 0.1 mL of water by gavage for 7 days before weaning, and then with 200 mg / L vancomycin in drinking water throughout the remainder of the sensitization protocol. Age- and sex-matched 3-week-old littermates were sensitized weekly by gavage with defatted, homemade peanut extract prepared from unsalted roasted peanuts (Hampton Farms, Severn, NC) and cholera toxin (CT) (List Biologicals, Campbell, CA) as previously described (see references B19 and B39; incorporated by reference in their entireties). Sensitization began at weaning and continued for 4 weeks. Before each sensitization, mice were fasted for 4–5 h and then given 200 μl of 0.2 M sodium bicarbonate to neutralize gastric acid. Thirty minutes later, mice were given 6 mg of peanut extract and 10 μg of cholera toxin (CT) in 150 μl of PBS by gavage.

[0179] Four weeks after sensitization, mice were rested for one week, after which a subset of mice was challenged with 1 mg of peanut extract in 200 μl of PBS via intraperitoneal (ip) administration. Rectal temperature was measured every 10 minutes immediately following challenge for up to 90 minutes using a rectal probe, and changes in core body temperature were recorded for each mouse. The remaining mice were not challenged and randomly assigned to experimental groups. In monotherapy experiments (Figure 16), one group of mice was treated with ButM by gavage twice daily for two weeks at 0.8 mg of total polymer per gram of mouse body weight (0.8 mg / g), while another group of mice received PBS. In dose-response studies (Figure 34), mice were treated twice daily with either PBS, ButM, or ButM at 0.8 mg / g (full dose), or 0.4 mg / g (half dose). Additionally, in an experiment in which ButM was delivered in synchronization with low-dose exposure to allergen (Figure 35), one group of mice was treated daily for 2 weeks with a low dose (200 μg) of peanut powder (PB2™ (PB2 Foods, Tifton, GA)), and another group of mice received both daily PB2™ (200 μg) and 0.8 mg / g ButM twice daily. After the treatment window, mice were challenged with 1 mg of peanut extract administered i.p., and core body temperature was measured for 90 minutes. Serum was collected from mice 90 minutes after challenge for measurement of mMCPT-1 and 24 hours after challenge for measurement of peanut-specific IgE. The collected blood was incubated at room temperature for 1 hour, centrifuged at 12,000 g for 7 minutes at room temperature, and serum was collected. - They were stored at 80°C and then analyzed. Serum antibodies and mMCPT-1 were measured by ELISA.

[0180] Measurement of mouse mast cell protease 1 (mMCPT-1) and serum peanut-specific IgE antibodies using ELISA mMCPT-1 was detected using an MCPT-1 mouse uncoated ELISA kit (Thermo Fisher) according to the protocol provided by the manufacturer. For peanut-specific IgE ELISA, serum from individual mice was added to peanut-coated Maxisorp Immunoplates (Nalge Nunc International, Naperville, IL). Peanut-specific IgE Abs were detected with unconjugated goat anti-mouse IgE (Southern Biotechnology Associates, Birmingham, AL) and rabbit anti-goat IgG-alkaline phosphatase (Invitrogen, Eugene, Oregon) and developed with p-nitrophenyl phosphate "PNNP" (SeraCare Life Sciences, Inc., Milford, MA). OD values ​​were converted to nanograms per milliliter of IgE by comparison with a standard curve of purified IgE by linear regression analysis and expressed as the mean concentration ± s.e.m. for each group of mice. Statistical differences in serum Ab levels were determined using a two-tailed Student's t-test. A P value <0.05 was considered significant.

[0181] 16S rRNA targeted sequencing Bacterial DNA was extracted using the QIAamp PowerFecal Pro DNA kit (Qiagen). Universal bacterial primers -563F (5'-nnnnnnnn-NNNNNNNNNNNN-AYTGGGYDTAAA-GNG-3') and 926R (5'-nnnnnnnn-NNNNNNNNNNNN-CCGTCAATTYHT-TTRAGT-3') (where "N" represents the barcode and "n" is an additional nucleotide added to the offset primer sequencing) were used to amplify the V4-V5 hypervariable region of the 16S rRNA gene from purified DNA. Illumina sequencing-compatible Unique Dual Index (UDI) adapters were ligated to the pools using the QIAsep 1-step Amplicon Library Kit (Qiagen). Library QC was performed using Qubit and Tapestation, followed by sequencing on the Illumina MiSeq platform at the Duchossois Family Institute Microbiome Metagenomics Facility at the University of Chicago. This platform generated 250-bp forward and reverse reads, which were analyzed for amplicon sequence variants (ASVs) using the Divisive Amplicon Denoising Algorithm (DADA2 v1.14) structure (Reference B56; incorporated by reference in its entirety). Taxonomy was assigned to the resulting ASVs using the Ribosomal Database Project (RDP) database with a minimum bootstrap score of 50 (Reference B57; incorporated by reference in its entirety). ASV tables, taxonomic classifications, and sample metadata were compiled using the phyloseq data structure (Reference B58; incorporated by reference in its entirety). Subsequent 16S rRNA relative abundance analysis and visualization were performed using R version 4.1.1 (R Development Core Team, Vienna, Austria).

[0182] Microbiome analysis To identify changes in the microbiome across conditions, linear discriminant analysis effect size (LEfSe) analysis was performed in R using the microbiomeMarker package and the run_lefse function (see references B59-B60; incorporated by reference in their entirety). Traits, specifically taxa, may or may not be associated with a given condition (e.g., after ButM treatment vs. PBS treatment), and effect sizes may be attributed to differences in taxa at selected taxonomic levels (LDA scores). In the LEfSe analysis, genera were compared as the main group. A significance level of 0.05 was selected for both the Kruskall-Wallis and Wilcoxon tests, and a linear discriminant analysis cutoff of 1.0 was implemented. The abundance of Clostridium cluster XIVa in post-treatment samples was also determined by quantitative PCR (qPCR) using the same DNA analyzed by 16S rRNA targeted sequencing. The commonly used primers 8F 61 and 338R 62 The total copy number of the 16S rRNA gene was quantified for normalization purposes using Clostridium cluster XIVa. 63 Primers specific for 2 were validated by PCR and qPCR. Primer sequences are listed in Table 1. qPCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems) according to the manufacturer's instructions. -CT The abundance of Clostridium cluster XIVa was calculated by the following formula, and all values ​​were within 1 (1 × 10 16 ) and expressed as a ratio of the total 16S copies per gram of intact fecal content. [Table 1]

[0183] toxicity research The toxic effects of pHPMA-b-pBMA on SPF C3H / HeJ mice were measured by hematological analysis using a Vet Axcel Chemistry Analyzer. Mice were treated with 0.8 mg / g body weight of NtL-ButM by daily gavage for 6 weeks. Weekly blood samples were obtained from each mouse and analyzed using a chemistry analyzer according to the manufacturer's instructions.

[0184] II. Results The copolymer formulates butyrate esters into water-suspendable micelles The amphiphilic block copolymer pHPMA-b-pBMA was synthesized through two steps of reversible addition-fragmentation chain transfer (RAFT) polymerization (Figure 12a). The hydrophilic block was formed from N-(2-hydroxypropyl)methacrylamide (HPMA), and the hydrophobic block was formed from N-(2-butanoyloxyethyl)methacrylamide (BMA), thus connecting the side chains of the backbone to butyrate esters via ester bonds. This ester bond can be hydrolyzed in the presence of esterase to release butyrate esters in the GI tract, resulting in a water-soluble polymer as the final product. In addition to pHPMA-b-pBMA, pMAA-b-pBMA was also synthesized, which has an anionic hydrophilic block formed from methacrylic acid (MAA) (Figure 12a). At the block size ratios used here, both pHPMA-b-pBMA and pMAA-b-pBMA contain 28 wt% butyrate esters.

[0185] These block copolymers can then be formulated into nanoscale micelles to achieve high suspension in aqueous solution and controlled release of butyrate esters from the core. pHPMA-b-pBMA was self-assembled into neutral micelles (NtL-ButM) through a cosolvent evaporation method (Figure 12b). The hydrophobic pBMA block forms the core, and the hydrophilic pHPMA forms the corona. In contrast, pMAA-b-pBMA cannot be formulated into micelles by this method due to the formation of intramolecular hydrogen bonds between pMAA chains (Reference B26; incorporated by reference in its entirety). However, titration of a strong base, here NaOH, into a mixture of pMAA-b-pBMA polymers can disrupt these bonds, converting methacrylic acid to ionized methacrylate esters (References B27-B29; incorporated by reference in their entireties). Upon base titration, the pMAA-b-pBMA polymer can then self-assemble into negatively charged micelles (Neg-ButM) (Figure 12b). Cryo-electron microscopy (cryoEM) revealed detailed structures of the micelles, especially the core structure formed by pBMA, which was more condensed with higher contrast. CryoEM images showed that the core diameter of NtL-ButM was 30 nm, while Neg-ButM had a smaller core diameter of 15 nm (Figures 12c and 12d). As measured by dynamic light scattering (DLS), both NtL-ButM and Neg-ButM have similar diameters of 44.7 ± 0.8 nm and 39.9 ± 1.6 nm, respectively (Figure 12e). Their low polydispersity index of less than 0.1 indicated the monodispersity of these micelles. NtL-ButM has a near-zero ζ-potential of -0.3 ± 0.5 mV, while Neg-ButM has a potential of -31.5 ± 2.3 mV due to the ionization of methacrylic acid (Figure 12e). To obtain the critical micelle concentrations (CMCs) of NtL-ButM and Neg-ButM, which indicate the possibility of micelle formation and dissociation in aqueous solution, pyrene was added during formulation, and the CMC was calculated by plotting the fluorescence intensity ratio between the first and third vibronic bands of pyrene. The results showed that Neg-ButM had a higher CMC of 14.0 ± 3.5 μM compared to that of NtL-ButM, which was 0.8 ± 0.3 μM (Figure 12e).The higher CMC indicated that Neg-ButM micelles dissociated more easily in solution, likely because the surface charge made the micellar structure less stable compared to the neutral micellar NtL-ButM. Additionally, small-angle X-ray scattering (SAXS) analysis was performed on both micelles to obtain the aggregation number (Figure 29). As shown by the Guinier plot, the radii of gyration for NtL-ButM and Neg-ButM were 14.2 nm and 13.5 nm, respectively (Figure 12e). The structure of the micelles was confirmed to be spherical by a Kratky plot of the SAXS data (Figure 29). The SAXS data were then fitted with a polydisperse core-shell spherical model (Reference B31; incorporated by reference in its entirety), assuming that the micelles have a spherical core with a higher scattering length density (SLD) and a shell with a lower SLD. This model provided the volume fraction of the micelles, the radius of the core, and the thickness of the shell, allowing the calculation of the aggregation number and the average distance between micelles. According to the fitting results, the aggregation numbers of NtL-ButM and Neg-ButM were 119 and 92, respectively (Fig. 12e).

[0186] Butyrate micelles release butyrate in the lower GI tract Considering that butyrate esters are linked to the micelle-forming chains via ester bonds, we examined the release of butyrate esters under ex vivo conditions, including simulated gastric and intestinal fluids, which mimic the biological environment. In simulated gastric fluid, both Neg-ButM and NtL-ButM showed a slight release of butyrate esters within a few hours and a sustained release over 3 weeks, although Neg-ButM had a slower release rate than NtL-ButM (Figure 13a). The anionic surface of Neg-ButM in an acidic environment likely contributes to the resistance of the BMA core to hydrolysis. In contrast, in simulated intestinal fluid, both micelles released most of the butyrate esters within minutes in the presence of a high concentration of the esterase pancreatin (Figure 13b).

[0187] After administering a single dose of NtL-ButM or Neg-ButM by gavage (ig), butyrate levels were measured in the mouse GI tract. Both LC-UV and LC-MS / MS methods have been used to measure butyrate concentrations in the luminal contents of the ileum, cecum, and colon, where butyrate-producing bacteria are commonly present (see References B32-B33; incorporated by reference in their entirety). However, because baseline concentrations in the ileum were too low for UV detection, LC-MS / MS was used to measure butyrate concentrations in that GI tract segment. NtL-ButM dramatically increased butyrate concentrations in the ileum for up to 2 h after gavage (Figure 13c), but this was short-lived, and butyrate concentrations did not increase in either the cecum or colon (Figures 13d and 13e). Neg-ButM increased butyrate concentrations in the cecum threefold, starting 4 h after gavage and sustained for at least an additional 8 h, but not in the ileum or colon (Figures 13c-13e). It is possible that butyrate released in the cecum continuously entered the colon, and the inability to detect an increase in butyrate concentrations in the colon may be due to its rapid absorption and metabolism by the colonic epithelium. In addition, the polymer backbone of the micelles remained intact during passage through the GI tract. Less than 28% molecular weight loss—as a percentage of butyrate content—of the polymer was observed in fecal samples collected 4–8 h after oral administration (Figures 31a and 31b). Furthermore, when incubated in an in vitro hydrolytic environment, the polymer backbone remained intact even after releasing the majority of the butyrate esters over a 7-day period in 125 mM sodium hydroxide solution.

[0188] By intravenously administering fluorescently labeled NtL-ButM or Neg-ButM to mice, we monitored the movement of micelles through the GI tract and visualized their biodistribution via an in vivo imaging system (IVIS) (Figure 32). A fluorescent marker was conjugated to the polymer chain, allowing visualization of the movement of the polymer backbone itself. IVIS results verified that the polymeric micelles were retained in the mouse GI tract for more than 6 hours after gavage. Neutral micellar NtL-ButM passed through the stomach and small intestine within 2 hours and accumulated in the cecum. However, negatively charged Neg-ButM initially accumulated in the stomach and then gradually migrated through the small intestine to the cecum. Overall, Neg-ButM had a longer retention time in the stomach and small intestine, which may be due to its stronger adhesive effect on the intestinal mucosa (see References B26, B34–B35; incorporated by reference in their entireties). Both micelles were excreted from the GI tract within 24 hours after administration. Additionally, the fluorescence signal in other major organs and plasma was measured by IVIS (Figure 32b), and the butyrate concentration in plasma was measured by LC-MS / MS. All signals were below the detection limit from both methods, indicating negligible absorption of these butyrate micelles from the intestine into the blood circulation, consistent with our desire to deliver butyrate to the GI tract and avoid the complications of systemic absorption of polymers or micelles.

[0189] Ileum-targeted butyrate micelles upregulate AMP genes in the ileal epithelium Delivery of butyrate ester to the lower GI tract may affect the host immune response by interacting with the intestinal epithelium. To investigate whether and how the butyrate ester micelles of the present invention regulate gene expression in the distal small intestine, we performed RNA sequencing of ileal epithelial cell compartments ( Figure 14a ). Germ-free (and therefore butyrate-depleted) C3H / HeN mice were treated ig with NtL-ButM daily for 1 week, and ileal epithelial cells were collected for RNA isolation and sequencing. Because only NtL-ButM (but not Neg-ButM) released butyrate ester in the ileum, we used only NtL-ButM in this experiment to examine its local effects. NtL-ButM-treated mice had a unique gene expression signature compared to mice treated with PBS or a control polymer consisting of the same polymer structure but without butyrate ester. These differences did not show gender dependence. Most of the genes upregulated by NtL-ButM treatment were Paneth cell-derived antimicrobial peptides (AMPs), including angiogenin 4 (Ang4), lysozyme-1 (Lyz1), intelectin (Itln1), and several defensins (e.g., Defa3, Defa22, and Defa24) (Figures 14a and 14c). The protein levels of intelectin, one of the upregulated AMPs, were quantified (Figures 14b and 14c). Intelectin is known to be expressed by Paneth cells, which reside in the small intestinal crypts and can recognize carbohydrate chains on bacterial cell walls (Reference B36; incorporated by reference in its entirety). Paneth cell AMPs are highly characteristic in C57BL / 6 mice, and specific reagents are available for their detection in this strain (Reference B37; incorporated by reference in its entirety). GF C57BL / 6 mice were gavaged with NtL-ButM or PBS daily for 1 week. Immunofluorescence microscopy of ileum sections revealed that the NtL-ButM-treated group expressed abundant intelectin in the crypts of the ileum tissue, whereas images from the PBS group showed limited intelectin signal (Fig. 14b).Quantification of the relative fluorescence intensity per ileal crypt using ImageJ also showed that the NtL-ButM group had significantly higher intelectin expression compared to the PBS control (Figure ​(Figure14c). 14c). Thus, intelectin staining further supported the pharmacological effect of NtL-ButM, and the upregulation of intelectin induced by NtL-ButM was not only demonstrated at the transcriptional level by RNAseq, but also verified at the protein level.

[0190] Butyrate micelles restore intestinal barrier function Butyrate-producing bacteria play an important role in maintaining the intestinal barrier. To evaluate the impact of locally delivered butyrate on intestinal barrier integrity, mice were treated with the chemical perturbant DSS for 7 days to induce epithelial barrier dysfunction (see reference B38; incorporated by reference in its entirety). Due to differences in the in vivo biodistribution and butyrate release behavior from the two butyrate micelles, it was reasoned that combined administration of NtL-ButM and Neg-ButM would cover the longest segment of the lower GI tract and persist for a longer period of time. Thus, a 1:1 combination of NtL-ButM and Neg-ButM (abbreviated as ButM) was selected for the study. Throughout DSS treatment and for 3 days after the end of DSS administration, mice were orally gavaged with either three different concentrations of PBS or ButM twice daily, or with cyclosporine A (CsA) once daily as a positive treatment control (as outlined in Figure 15a). Intestinal barrier permeability was assessed using gavage of 4 kDa FITC-dextran. Significantly higher concentrations of FITC-dextran were detected in the serum of DSS-treated mice gavaged with PBS alone, demonstrating intestinal barrier damage. Naive mice (without DSS exposure) or DSS-treated mice that also received either CsA or ButM at all three concentrations had similar serum levels of FITC-dextran, indicating that treatment with ButM successfully repaired DSS-induced barrier damage (Figure 15b). In addition, neonatal antibiotic treatment impairs homeostatic epithelial barrier function and increases permeability to food antigens (Reference B19; incorporated by reference in its entirety). Therefore, we further evaluated whether ButM treatment could reduce intestinal barrier permeability in antibiotic-treated mice (Figure 15c). Similar to what was observed in the DSS-induced model, mice treated with ButM had significantly lower FITC-dextran levels in serum compared with mice fed PBS (Fig. 15d), demonstrating that ButM effectively rescued both DSS- and antibiotic-induced intestinal barrier dysfunction.

[0191] Butyrate micelles ameliorated anaphylactic responses in peanut-allergic mice To evaluate the efficacy of butyrate-containing micelles in treating food allergies, ButM was tested in a well-established mouse model of peanut-induced anaphylaxis (References B19, B39; incorporated by reference in their entireties). All mice were treated with vancomycin to induce dysbiosis. Starting from weaning, vancomycin-treated SPF C3H / HeN mice were intragastrically sensitized weekly for 4 weeks with peanut extract (PN) plus the mucosal adjuvant cholera toxin (CT) (Figure 16a, b), as previously described (References B19, B39; incorporated by reference in their entireties). After sensitization, some mice were challenged intraperitoneally (ip). To ensure uniform sensitization of mice, changes in PN and core body temperature were monitored; a decrease in core body temperature indicates anaphylaxis (Figure 16c). The remaining sensitized mice were then treated i.g. twice daily for two weeks with either PBS or the combined micellar formulation ButM. After two weeks of treatment, the mice were challenged by i.p. injection of PN, and core body temperature was assessed to evaluate their response to allergen challenge. Compared to PBS-treated mice, allergic mice treated with ButM experienced a significantly reduced anaphylactic reduction in core body temperature (Figure 16d). In addition, ButM-treated mice also had significantly reduced levels of mouse mast cell protease-1 (mMCPT-1) and peanut-specific IgE detected in their serum (Figures 16e and 16f). mMCPT-1 is a chmyase expressed by intestinal mucosal mast cells, and increased levels of mMCPT-1 increase in intestinal barrier permeability during allergic hypersensitivity responses (References B40-B41, incorporated by reference in their entireties). Furthermore, these effects of ButM on peanut-allergic mice were dose-dependent, as reducing the dose of ButM by half was observed to be less effective than the full dose in protecting mice from anaphylactic responses. Collectively, these results demonstrate that ButM as monotherapy can effectively prevent allergic responses to food in sensitized mice.

[0192] Because OIT is the only FDA-approved treatment for peanut allergy, we tested whether ButM is an effective treatment when delivered synchronously with low-dose exposure to allergen in sensitized mice (Figures 35a and 35b). Low-dose PN treatment alone in this regimen had no therapeutic effect, likely due to an insufficient length of treatment to achieve functional OIT, as low-dose PN-treated mice had a similar decrease in core body temperature to untreated mice (Figures 35c and 35d). However, mice treated with low-dose PN plus ButM exhibited a significantly reduced decrease in core body temperature, indicating a substantially reduced anaphylactic response. This suggests the potential clinical use of butyrate micelles for patients undergoing OIT. However, the treatment did not reduce serum peanut-specific IgE, as has been observed in several clinical studies of OIT (References B8 and B43; incorporated by reference in their entireties) (Figure 35f).

[0193] Butyrate micelles alter fecal microbiota and promote clostridia recovery after antibiotic exposure Given that ButM can induce AMPs and alter gut metabolism, we investigated whether treatment alters the fecal microbiota. In the mouse model of peanut allergy described above, we induced dysbiosis by treating mice with vancomycin 1 week before the onset of allergen sensitization and throughout the sensitization regimen. Vancomycin depletes Gram-positive bacteria, including Clostridium species (see Reference B43, incorporated by reference in its entirety). After sensitization, vancomycin was removed from the drinking water, and the fecal microbial composition of allergic mice was compared before and after treatment with PBS or ButM (see collected time points in Figure 16a). 16S rRNA targeted sequencing confirmed the depletion of Clostridium in vancomycin-treated mice; instead, the fecal microbiota was dominated by Lactobacillus and Proteobacteria (Figure 17a, left). After vancomycin administration was discontinued, regrowth of Clostridia (including Lachnospiraceae and others) and Bacteroidetes was observed in both the PBS- and ButM-treated groups (Figure 17a, right, Figure 36). Comparing differentially abundant taxa between treatment groups by LEfSe analysis, Murimonas and Streptococcus were significantly more abundant in the PBS-treated group compared with the ButM-treated group (Figure 17b). ButM treatment significantly increased the relative abundance of Enterococcus, Coprobacter, and Clostridium cluster XIVa (Figure 17b). Clostridium cluster XIVa is a numerically predominant bacterial group known to produce butyrate esters (in both mice and humans), modulate host immunity, and induce Tregs (References B43–B44; incorporated by reference in their entireties). The relative abundance of Clostridium cluster XIVa in ButM-treated mice was significantly increased in the 16S dataset (Fig. 17c), and the enrichment in the abundance of this taxon was quantified by qPCR (Fig. 17d).The finding of increased abundance of Clostridium cluster XIVa after treatment with ButM is consistent with previous work showing that butyrate sensing by peroxisome proliferator-activated receptor (PPAR-γ) switches colonocyte metabolism toward β-oxidation, creating a local hypoxic niche for these oxygen-sensitive anaerobes (ref. B45; incorporated by reference in its entirety).

[0194] Characterization of the toxicity of butyrate micelles demonstrated that treatment did not induce changes among the serological toxicity markers tested, including serum albumin, alanine aminotransferase, amylase, blood urea nitrogen, calcium, and total protein, over 6 weeks of daily treatment (Figure 37).

[0195] References The following references, some of which are cited above, are incorporated herein by reference in their entirety: A1. Chang, PV;Hao, L.;Offermanns, S.;Medzhitov, R., The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition. Proceedings of the National Academy of Sciences 2014, 111 (6), 2247. A2. Mathewson, ND;Jenq, R.;Mathew, AV;Koenigsknecht, M.;Hanash, A.;Toubai, T.;Oravecz-Wilson, K.;Wu, S.-R.;Sun, Y.;Rossi, C.;Fujiwara, H.;Byun, J.;Shono, Y.;Lindemans, C.;Calafiore, M.;Schmidt, TM;Honda, K.;Young, VB;Pennathur, S.;van den Brink, M.;Reddy, P., Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease. Nature Immunology 2016, 17 (5), 505-513. A3. Fachi,JL;Felipe,JS;Pral, LP;da Silva, BK;Correa, RO;de Andrade, MCP;da Fonseca, DM;Basso, PJ;Camara, NOS;de Sales, ESE;Dos Santos Martins, F.;Guima, SES;Thomas, AM;Setubal,JC;Magalhaes, YT;Forti, FL;Candreva, T.;Rodrigues, HG;de Jesus, MB;Consonni, SR;Farias, ADS;Varga-Weisz, P.;Vinolo, MAR, Butyrate Protects Mice from Clostridium difficile-Induced Colitis through an HIF-1-Dependent Mechanism. Cell Rep 2019, 27 (3), 750-761.e7. A4 Arpaia, N.;Campbell, C.;Fan, X.;Dikiy, S.;van der Veeken,J.;deRoos,P.;Liu,H.;Cross,JR;Pfeffer,K;Coffer,PJ;Rudensky,AY, Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 2013, 504(7480), 451–455. 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E., Structure of Micelles of Poly(n-butyl acrylate)-block-poly(acrylic acid) Diblock Copolymers in Aqueous Solution. Macromolecules 2007, 40 (12), 4351-4362. A9. Colombani, O.;Ruppel, M.;Schubert, F.;Zettl, H.;Pergushov, D. V.;Muller, A. H. E., Synthesis of Poly(n-butyl acrylate)-block-poly(acrylic acid) Diblock Copolymers by ATRP and Their Micellization in Water. Macromolecules 2007, 40 (12), 4338-4350. A10. Xu, F.;Xu,J.W.;Luo, Y. L., Impact of hydrogenation on physicochemical and biomedical properties of pH-sensitive PMAA-b-HTPB-b-PMAA triblock copolymer drug carriers. J Biomater Appl 2016, 30 (10), 1473-84. A11. Felber, A. E.;Dufresne, M.-H.;Leroux, J.-C., pH-sensitive vesicles, polymeric micelles, and nanospheres prepared with polycarboxylates. Advanced Drug Delivery Reviews 2012, 64 (11), 979-992. A12. Lele, B. S.;Hoffman, A. S., Mucoadhesive drug carriers based on complexes of poly(acrylic acid) and PEGylated drugs having hydrolysable PEG-anhydride-drug linkages. 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Claims

1. A composition comprising micelles of a copolymer of methacrylic acid (MAA) and N-(2-alkanoyloxyethyl) methacrylamide (AMA).

2. The composition of claim 1, wherein the copolymer is a block copolymer having the following structure: (a) 【Chemistry 1】 wherein a and b are independently 1 to 1000; (b) 【Chemistry 2】 wherein a and b are independently 1 to 1000; (c) 【Transformation 3】 wherein a and b are independently 1 to 1000; (d) 【Chemistry 4】 wherein a and b are independently 1 to 1000; (e) 【Transformation 5】 wherein a and b are independently 1 to 1000; or (f) 【Transformation 6】 In the formula, a and b are independently 1 to 1,000.

3. The copolymer is a random copolymer having the following structure: 【Transformation 7】 wherein each Y is independently: (a) 【Transformation 8】 ： (b) 【Chemistry 9】 ： (c) 【Chemistry 10】 ： (d) 【Chemistry 11】 ： (e) 【Chemistry 12】 or (f) 【Chemistry 13】 The composition of claim 1 selected from the group consisting of:

4. The composition of claim 1, comprising a second micelle of a second copolymer of 2-hydroxypropyl methacrylamide (HPMA) and N-(2-alkanoyloxyethyl) methacrylamide (AMA).

5. The composition of claim 4, wherein the second copolymer is a block copolymer having the following structure: (a) 【Chemistry 14】 wherein a and b are independently 1 to 1000; (b) 【Chemistry 15】 wherein a and b are independently 1 to 1000; (c) 【Chemistry 16】 wherein a and b are independently 1 to 1000; (d) 【Chemistry 17】 wherein a and b are independently 1 to 1000; (e) [Chemistry 18] wherein a and b are independently 1 to 1000; or (f) 【Chemistry 19】 In the formula, a and b are independently 1 to 1,000.

6. The copolymer is a random copolymer having the following structure: 【Chemistry 20】 wherein each Y is independently: (a) 【Chemistry 21】 ： (b) 【Chemistry 22】 ： (c) 【Chemistry 23】 ： (d) 【Chemistry 24】 ： (e) 【Chemistry 25】 or (f) 【Chemistry 26】 The composition of claim 4 selected from the group consisting of:

Images