Metabolite-based polymers and nanoparticles for the treatment of inflammatory diseases

Abstract

Cargo-free metabolite-based nanoparticles with high biocompatibility and low organ toxicity are provided to dampen pro-inflammatory responses while mitigating accompanying metabolic dysregulation in certain inflammatory conditions such as obesity and its metabolic sequelae such as type 2 diabetes. These nanoparticles are also useful as treatment for inflammatory and allergic conditions such as allergic airway inflammation. These nanoparticles are engineered from biodegradable polymers comprising endogenous bioactive metabolites, which upon cellular internalization are degraded into their monomeric components over time to elicit multimodal immunomodulatory functions without the need for frequent dosing, including inducing anti-inflammatory immune cell phenotypes, i.e., M2 macrophages.

Description

METABOLITE-BASED POLYMERS AND NANOPARTICLES FORTHE TREATMENT OF INFLAMMATORY DISEASESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international (PCT) application, which claims the benefit of United States provisional application serial no. 63 / 611,786, filed 19 December 2023. The entire contents of the aforementioned provisional application is hereby incorporated by reference as if fully set forth herein.GOVERNMENT FUNDING SUPPORT

[0002] This invention was made with government support under Grant Numbers GM142752 and TR003098 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] 1. Field of the InventionThe invention relates to the general field of medicine. In particular, the invention is a platform technology for the treatment of inflammatory diseases and conditions including obesity, allergy, sepsis, infections disease, autoimmunity, spinal cord injury, cancer, and many others.

[0004] 2. Background of the invention

[0005] Itaconate (ITA) is a small unsaturated dicarboxylic acid that is a key metabolite of activated macrophages and is the product of the mitochondrial enzyme cis-aconitate decarboxylase (ACOD1). Itaconate is well known to possess immunomodulatory and cytoprotective properties in inflammatory and infectious diseases. Chemically modified derivatives of ITA such as dimethyl- and 4-octyl-itaconate (4-OI) are being investigated therapeutically to treat those diseases. Despite the extensive research having been performed using ITA, the immunomodulatory properties and corresponding therapeutic utility of its naturally occurring isomers, mesaconate (MSA) and citraconate (CTA) are not well established. Notably, the small molecule metabolites noted are not cell membrane permeable and thechemically modified derivative of ITA, 4-01, has been demonstrated to not function through the same molecular mechanism. Thus, there is a need to develop novel strategics to enable the functional delivery of these endogenous metabolites to cells of interest to exert immunomodulatory and therapeutic functions.

[0006] The World Health Organization (WHO) states that more than two billion people worldwide are obese, and that number is rapidly growing. In 2022, 1 in 8 people worldwide were living with obesity; 2.5 billion adults 18 years and older were overweight, and 890 million were obese. Approximately 167 million new adults and children are predicted to become obese and less healthy by the end of 2025. It is causing critical concerns worldwide, not only because it affects a person's appearance but also because it is associated with several comorbidities, including cardiovascular disease, and type II diabetes.

[0007] The consequences of obesity are of critical concern due to its wide-reaching associations with metabolic diseases, including type II diabetes, metabolic dysfunction-associated steatohepatitis (MASH, formerly known as NASH), metabolic dysfunction-associated steatotic liver disease (MASLD, formerly known as non-alcoholic fatty liver disease (NAFLD)), cardiovascular disease, and cancer, as well as increased aggregate medical costs of over $100B.

[0008] Obesity can be characterized as a chronic relapsing disease with a multifactorial pathology and a chronic, low-grade inflammation and metabolic dysfunction influenced by genetics, nutrition, lifestyle, and environmental factors. Excess triglycerides created and deposited in fat cells contribute to an imbalance in energy intake and expenditure by adipose cells. As adipocytes in adipose tissue (AT) enlarge, they can become hypoxic and dysfunctional, triggering the release of soluble factors that recruit various immune cells like macrophages, among others. These immune cells and adipocytes secrete pro-inflammatory cytokines, driving chronic inflammation, and promoting insulin resistance. AT serves as both an energy storage depot and a secretory organ, producing a range of bioactive substances that play a crucial role in metabolic homeostasis. These substances include important hormones such as leptin, visfatin, and adiponectin.

[0009] AT, as a source of inflammatory mediators, can produce cytokines such as tumor necrosis factor TNFa and interleukin-6 (IL-6). These inflammatory mediators' excessive production and release are associated with insulin resistance, atherosclerosis, and other illnesses.Obesity (along with its associated metabolic dysregulation) is one condition where inflammatory processes serve an important role.

[0010] Macrophages are innate immune cells that play a central role in host defense, wound healing, and immune regulation. Although macrophage phenotypes exist on a spectrum, they can generally be classified as pro-inflammatory, Ml-like (Ml), or anti-inflammatory, M2-like (M2). In lean individuals, AT macrophages (ATMs) in brown adipose tissue (BAT) predominantly exhibit an M2 phenotype. However, during obesity, ATMs are activated in response to excessive AT expansion, which initiates pro-inflammatory signaling, shifting the balance from 4:1 to 1.2:1 M2-to-Ml ratio, while enhancing the trafficking of additional inflammatory immune cells into AT.

[0011] ATMs play a critical role in the regulation of inflammation in AT. During obesity, AT undergoes a significant expansion that leads to the recrutiment of many immune cells including ATMs, which become activated in response to signals from adipocytes as well as other environmental cues altering their phenotype towards a classically-activated Ml state. The activation of ATMs and subsequent secretion of various pro-inflammatory cytokines such as IL- 6, TNFa, and IL-ip can interfere with insulin action in adipocytes. The skewing of macrophage polarization contributes to the transformation of BAT into white adipose tissue (WAT), a hallmark of obesity. Thus, obesity is associated with an imbalance in the ratio of M2 / M1 macrophages within AT.

[0012] Current obesity treatments can be broadly categorized into lifestyle modifications and medical interventions, including surgery and pharmacotherapy, with multiple drugs being Food and Drug Administration (FDA)-approved for chronic weight management such as lipase inhibitors (XenicalIM), GLP-1 and / or GIP receptor agonists (Saxenda1M, Wegovy1M, Zepbound™), melanocortin 4 receptor agonists (IMCIVREE). Additional approved combination therapies alter the brain’s sense of reward (Contrave™) or act to decrease appetite (Qsymia™).

[0013] While current treatments for obesity, such as weight-loss medications, surgery, dietary modifications, and exercise programs, have shown promising results, it is important to acknowledge that these treatments often involve frequent dosing requirements, high cost, nonresponder issues, and potential for serious side effects. Potential side effects can include pancreatitis, liver injury, bone / muscle loss, heart complications, and others.

[0014] For example, prescription weight-loss medications such as Orlistat™ may cause gastrointestinal side effects such as oily stools, flatulence, abdominal pain, diarrhea, bladder pain, body aches, difficulty with breathing, ear- congestion, general feeling of discomfort or illness, loss of voice, muscle aches and pains, nasal congestion, and in some cases death. Other medications, including appetite suppressants, may cause side effects like increased heart rate, elevated blood pressure, insomnia, dry mouth, or mood changes.

[0015] Surgical procedures are another type of treatment, including procedures bariatric surgery like gastric bypass or gastric sleeve surgery, which can be effective for weight loss in certain individuals. However, any surgery carries risks, and potential side effects can include infections, bleeding, blood clots, hernias, dumping syndrome (rapid emptying of stomach contents into the small intestine), nutrient deficiencies, gallstones, and gastrointestinal issues. Long-term followup and adherence to post-surgical dietary and lifestyle recommendations are necessary for such a treatment. Because WAT is so important to metabolism, the removal of AT through surgical procedures like liposuction (surgical fat removal) can have significant implications for overall health. Due to the potential impact on metabolic balance and the disruption of the beneficial functions of AT, liposuction is not a preferred approach for obesity.

[0016] Dietary modifications are frequently employed as a treatment for obesity. However, it is crucial to consider that implementing dietary changes, such as following reduced-calorie or specialized diet plans, can lead to side effects depending on the approach and individual response. Rapid weight loss diets or very low-calorie diets, if not properly managed, may result in fatigue, nutrient deficiencies, constipation, electrolyte imbalances, and even potentially fatal consequences. Exercise is one of the safest treatments for obesity, however it is not effective for all types of obesity, and a clinical study showed that weight loss induced by exercise was only modest.

[0017] Nearly one-third of Americans suffer from allergies, experiencing symptoms ranging from itchy eyes to life-threatening anaphylaxis, negatively impacting their quality of life. Despite this significant burden, current therapeutic guidelines rely on symptom management rather than a curative approach. Current therapies include corticosteroids, antihistamines, bronchodilators, and leukotriene inhibitors, among others.

[0018] To address the underlying pathology, allergen- specific immunotherapy (SIT) aims to promote T cell differentiation towards Thl or T regulatory cell (Tregs) responses, therebycounterbalancing the Th2 response and desensitizing patients to allergens. However, administering soluble antigen poses a risk of anaphylaxis due to immune recognition and prolonged and repeated dosing, necessitating close monitoring and co-administration of therapies like omalizumab (anti-IgE) to minimize systemic allergic reactions.

[0019] Activation of antigen presenting cells (APCs) by foreign antigens (Ag) results in key metabolic shifts, including dysregulation in the tricarboxylic acid (TCA) cycle. This disruption in the TCA cycle promotes glycolysis and induces oxidative stress. Specifically, immune response gene 1 (,Irgl a gene encoding for the enzyme czs-aconitate decarboxylase (ACOD1), is upregulated and catalyzes production of the immunomodulatory metabolite itaconate (ITA). Although endogenous levels of ITA increase during AAD, this rise is not sufficient to counteract inflammation. During allergic airway disease (AAD), Irgl expression is upregulated in APCs like alveolar macrophages and dendritic cells. ITA exerts its anti-inflammatory and anti-oxidant effects by inhibiting succinate dehydrogenase (SDH), activating nuclear factor erythroid 2- related factor 2 (Nrf2), and blocking aerobic glycolysis (key contributors to allergy-related inflammation). Mesaconate (MSA), a non-Michael acceptor isomer derived directly from ITA, reduces pro-inflammatory responses through distinct non-covalent mechanisms with potentially fewer metabolic disruptions.

[0020] Pulmonary macrophages or alveolar macrophages (AMs) are the first line of defense for invading pathogens and antigens to the lung and demonstrate similar macrophage skewing depending on foreign particulate. Furthermore, AMs release chemokines and cytokines responsible for recruitment of innate and adaptive immune cells, thus providing crucial support to the lung for tissue repair and homeostasis during AAD. A study demonstrated that Irgl deficient dendritic cells have enhanced antigen presentation to T cells thus accelerating Th2 effector functions. Providing exogenous ITA as a cell membrane-permeable derivative, 4-octyl itaconate (4-OI), reduced IgE and Th2 responses in murine models of house dust mite (HDM)- and ovalbumin (0VA) / Alum- induced AAD when administered daily and prior to each allergen challenge. The mechanisms behind metabolic reprogramming of the innate and adaptive immune cell responses in AAD are not well understood.

[0021] It has become apparent that the Irgl / itaconate axis is involved in AAD. Polymer-based nanoparticles (NPs) have been employed as an antigen delivery system to induce tolerance and in some allergic disease models. For example, ovalbumin (OVA) encapsulated poly(lactic-co-glycolic acid) (PLGA) NPs can attenuate antigen-specific IgE and Th2 associated cytokines when administered prophylactically in an OVA / Alum- A AD model, however, when administered therapeutically, it failed to inhibit antigen specific IgE and partially reduced Th2 cytokines. While PLGA is widely studied and ideal due to its biocompatibility and biodegradability, its degradation products lactic acid and glycolic acid are excreted and do not address the underlying metabolic dysregulation driving Th2 inflammation in AAD.

[0022] Thus, there is a critical need for an improved therapeutic strategy that induces Th2 tolerance, while mitigating the risk of hypersensitivity.

[0023] Inflammatory conditions in general are an important class of diseases and conditions affecting large portions of the population. In particular, obesity and its sequelae are an important group of diseases and conditions which have been difficult to address. As well, treatment for allergies have been generally based on symptom management only. Therefore, identifying novel treatments for obesity and allergy can improve long-term health and well-being while reducing the risk of other chronic conditions, leading to advances in patient outcomes.SUMMARY OF THE INVENTION

[0024] Thus, there exists a critical need in the art for new effective and safe therapies, especially natural products, with potential anti-inflammatory, antibacterial, and anti-obesity activity, as well as the ability to skew macrophage polarization away from inflammatory M 1 towards regenerative and anti-inflammatory M2 phenotypes for use in wound healing. Mitochondrial metabolism and the production of metabolites have been identified as crucial regulators of inflammatory signaling and macrophage phenotypes. One such metabolite is Itaconate (ITA), derived from the tricarboxylic acid (TCA) cycle through the enzyme cis-aconitate decarboxylase (ACOD1 / IRG1). ACOD1 and ITA are highly upregulated genes and metabolites during pro- inflammatory macrophage activation. A recently discovered function of ITA and its isomers, mesaconate (MSA), and citraconate (CTA), are their differential activities for macrophage immunomodulation, where all three isomers inhibited glycolysis to a similar extent, altered amino acid metabolism, modulated cytokine / chemokine release, reduced interferon signaling, and oxidative stress.

[0025] Specifically, the invention here provides a polymer comprising or consisting essentially of itaconatc (ITA), mcsaconatc (MSA), or citraconatc (CTA). Preferably, the polymer is biodegradable.

[0026] In certain embodiments, the invention provides a nanoparticle comprising a polymer as mentioned above. The invention provides a nanoparticle comprising a pharmaceutical composition selected from the group consisting of an ITA polymer, an MSA polymer, and a CTA polymer.

[0027] In additional embodiments, the invention provides a pharmaceutical composition containing the nanoparticle described above.

[0028] hi certain embodiments, the invention relates to a method of treating an inflammatory condition, traumatic injury, or infectious disease in a subject in need thereof, comprising administering to the subject the polymers described herein or the nanoparticles described herein to a subject. Preferably, administration of the polymer or the nanoparticle to a subject induces CD206 in macrophages or immune cells of the subject. Preferably the inflammatory condition is selected from the group consisting of cancer, obesity, infectious disease, sepsis, allergic airway disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis, diabetes, lupus, multiple sclerosis, cardiovascular disease, fibrosis and autoimmune diseases. Most preferably, the inflammatory condition is obesity, allergic airway disease, or sepsis.

[0029] In other embodiments, the invention relates to methods wherein the infectious disease is selected from the group consisting of a bacterial, viral, or parasitic infection. The infectious disease can be caused by a Gram-positive bacterium or a Gram-Negative bacterium, or by COVID-19.

[0030] In other embodiments, the invention relates to a method of inducing an antiinflammatory response in a subject suffering from an inflammatory condition comprising administering to the subject the nanoparticle of the invention to the subject. Preferably, this subject suffers from obesity, allergic airway disease, or sepsis.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Certain embodiments are illustrated by way of example, and not by way of limitation in the figures of the accompanying drawings.

[0032] FIG. 1 is a schematic overview of metabolite-based nanoparticle treatment of obesity.

[0033] FIG. 2A and FIG. 2B present data on the change in diameter and the change in zeta potential, respectively, upon storage at the noted temperatures.

[0034] FIG. 3A and FIG. 3B present data on nanoparticle tracking analysis.

[0035] FIG. 4 shows the results of a time- and pH-dependent degradation study.

[0036] FIG. 5A, FIG. 5B shows particle uptake in M0 and Ml BMM0s over 3 hours for confocal microscopy and 24 hours for flow cytometry.

[0037] FIG. 6 presents TOM20 staining and shows that pITA-NP treatment reduces mitochondrial length, suggesting an altered cellular phenotype.

[0038] FIG. 7 shows the effects of polymer (pITA) molecular weight, subsequent pITA-NP formation, and treatment of M0, Ml, and M2 bone marrow-derived macrophages.

[0039] FIG. 8 presents data on the effects of 4-OI, DMI, ITA, and pITAjiigh-NP on the phenotypes of M0 bone marrow-derived macrophages.

[0040] FIG. 9 presents data on the effects of 4-OI, DMI, ITA, and pITAuigh-NP on the phenotypes of Ml bone marrow-derived macrophages.

[0041] FIG. 10A, FIG. 10B, and FIG. 10C show flow cytometry results. The tests were performed to evaluate M0 and Ml BMM0 polarization following particle treatment at 3, 8 and 24 hours (30 pg / mL concentration).

[0042] FIG. 11 provides histograms of live, F4 / 80+and CD1 lb+cell surface molecule expression with the red line histograms representing the treatment groups while the grey filled histograms represent the untreated BMM0S.

[0043] FIG. 12A through FIG. 12E present data showing that macrophages displayed altered surface molecule expression when treated with higher concentrations of NPs. Statistical differences were determined by one-way ANOVA with Tukey’s multiple comparisons test. Within data subsets, no significant difference was observed between conditions labeled with matching letters (p>0.05). Error bars indicate SD.

[0044] FIG. 13A presents viability data indicated by flow cytometry. FIG. 13B and FIG. 13C show the results for analysis of IL-6 and MCP-1 secretions using ELISA, respectively.

[0045] FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D show the effects of particle treatment of M0 bone marrow-derived macrophages.

[0046] FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D show the effects of particle treatment of Ml bone marrow-derived macrophages.

[0047] FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D show the effects of particle treatment of M2 bone marrow-derived macrophages.

[0048] FIG. 17A, FIG. 17B, and FIG. 17C present ALT, AST, and creatinine kinase, respectively as a test of in vivo toxicity.

[0049] FIG. 18A is a brief timeline of the therapeutic strategy for induction of allergic airway disease and particle treatment of 5-7 week old female BALB / c mice.

[0050] FIG. 18B shows total IgE in plasma showed a significant reduction with mice treated with a single dose of pITA-NP and pMSA-NP on day 14. Sample collection occurred on day 18.

[0051] FIG. 18C, FIG. 18D, FIG. 18E, and FIG. 18F show a significant reduction of Th2 associated cytokines IL-4 (FIG. 18C), IL-5 (FIG. 18D), IL-13 (FIG. 18E), and IFNy (FIG. 18F). Sample collection occurred on day 18.

[0052] FIG. 19A through FIG. 19F are photomicrographs showing macrophages collected from lung lavage. Sample collection occurred on day 18.

[0053] FIG. 20A and FIG. 20B show average eosinophil and macrophage counts, respectively.

[0054] FIG. 21A and FIG. 21B are a schematic and set of photographs, respectively showing 3T3- LI adipocytes were seeded and differentiated into mature adipocytes, then cultured with LPS stimulated macrophages on a transwell insert for 24 hours, then treated with 20 pg / mL nanoparticles for 24, 48 and 72 hours.

[0055] FIG. 21C, FIG. 21D, and FIG. 21E show data for the cells from FIG. 20B, tested for the ability of NPS to alter adipose area (FIG. 21C), integrated optical density (FIG. 21D) and Feret’s Diameter (FIG. 21E) using ImageJ™.

[0056] FIG. 22A and FIG. 22B are a schematic and graph showing normalized body weight in HFD-induced obese mice after 2 mg / dose of pITAuigh-NP, 2 mg / dose PLGA-NP, or PBS control was injected 3 times subcutaneously to ING and AXI adipose tissue, respectively.

[0057] FIG. 23 is a graph showing the fold change in weight in the high fat diet-induced obesity mouse model to evaluate the effects of pMSAuow-NP and pCTA ow-NP on body weight.

[0058] FIG. 24A is a set of photographs showing tissues after treatment and collection.

[0059] FIG. 24B shows data for tissue weight from the mice of FIG. 24A collected on day 30.

[0060] FIG. 25 shows representative images and H&E staining of livers from mice of FIG. 24A.

[0061] FIG. 26 presents data on blood cytokine analysis to assess IL-6, IL- 1 , TNF-a, IL- 10, IL- 2, IL-4, CRP, T3, and TSH levels.

[0062] FIG. 27 A and FIG. 27B are sets of photographs showing H&E staining performed on the visceral fat (FIG. 27 A) and the fat surrounding the kidneys (FIG. 27B) to evaluate adipocyte size.

[0063] FIG. 28 shows additional H&E staining was on heart, lung, liver, spleen and kidneys.

[0064] FIG. 29A, FIG. 29B, and FIG. 29C show the total area, Feret’s diameter, and integrated optical density, respectively, calculated from the visceral adipose tissue.

[0065] FIG. 30 presents flow cytometry data on the adipose tissue to assess UCP1 expression in inguinal, axillary, and visceral fat pads, as indicated, to determine the phenotype.

[0066] FIG. 31 shows hierarchical clustering of gene expression in AXI, ING, VIS, and liver tissues showing induction of genes associated with fat browning / thermogenesis and insulin sensitivity are enhanced after pITA-NP treatment.

[0067] FIG. 32 shows Principal component analysis (PCA) of Ctrl (obese control), PLGA-NP, and pITA-NP treatments revealed distinct clustering of pITA-NP from the other groups.

[0068] FIG. 33A and FIG. 33B show that Ucpl (FIG. 33A) and Adiponectin (FIG. 3B) expression is significantly upregulated in multiple tissues.

[0069] FIG. 34A, FIG. 34B, FIG. 34C, and FIG. 34D are the results of an LPS-induced endotoxemia survival study to evaluate the efficacy of metabolite-based nanoparticles.

[0070] FIG. 35 A, FIG. 35B, FIG. 35C are bar graphs presenting data for activity of metabolites against E. coli, and 5. aureus, respectively.

[0071] FIG. 36 is a growth curve (OD600 Values) over a 24-hour period in UPEC. Soluble metabolites were not significantly effective in reducing OD600 values but had minor decrease post- stationary phase.

[0072] FIG. 37 is a growth curve for S. aureus after the indicated treatments.

[0073] FIG. 38 is a bar graph showing that concentration-dependent effects of particles on PAO1 CFUs. The figure shows %Control CFU in PAO1.

[0074] FIG. 39A and FIG. 39B are bar graphs showing CFU raw and normalized data for particles treated at 1 mg / mL (PAO1).

[0075] FIG. 40A and FIG. 40B are bar graphs showing CFU raw and normalized data for particles treated at 1 mg / mL (UPEC).

[0076] FIG. 41A and FIG. 41B are bar graphs showing CFU raw and normalized data for particles in N2.DETAILED DESCRIPTION OF THE INVENTION

[0077] 1. Overview

[0078] Adipose tissue macrophages (ATMs) contribute to fat development: recent studies have highlighted the potential benefits of immunomodulation for the prevention and treatment of obesity and its associated metabolic dysregulation. Macrophages are innate immune cells that play a central role in responding to pathogens, initiating and controlling inflammation, and aiding in tissue repair. Research has demonstrated the remarkable plasticity of macrophages to dynamically alter their polarization between pro-inflammatory, Ml-like (Ml), and antiinflammatory, M2-like (M2) states in response to environmental cues.

[0079] ATMs, initially present in lean, visceral white adipose tissue (WAT) depots, are recruited and undergo activation and pro-inflammatory signaling in response to excessive adipose tissue (AT) expansion during obesity. The polarization state of macrophages contributes to the transformation of brown adipose tissue (BAT) into WAT, which is a hallmark of obesity. The expansion of AT is closely linked to the ratio of M2 and Ml macrophages present. During obesity, ATMs are activated in response to excessive AT expansion, which initiates pro- inflammatory signaling, shifting the balance of M2-to-Ml macrophages from 4:1 to approximately 1.2:1, while enhancing the trafficking of additional immune cells into fat tissue. In obese individuals, Ml macrophages are typically increased, while M2 macrophages are reduced.

[0080] Macrophage modulation has been suggested as an effective therapeutic approach for preventing and treating different diseases, including obesity, and we have recently demonstrated this concept by developing simvastatin-encapsulated nanoparticles (Sim-NP) for obesity treatment, where we found that Sim-NP repolarized Ml macrophages towards M2 in AT, reduced inflammation, converted WAT to BAT (beiging), and induced weight loss in obese mice. Thus, developing strategies to safely and effectively correct this imbalance by promoting M2 macrophage polarization may offer significant potential to resolve obesity.

[0081] AT plays a role as a metabolic tissue driving inflammatory responses in obesity. The relationship between metabolic regulation, immune responses during obesity, and AT inflammation is intricately intertwined. Extensive research has demonstrated that pro-inflammatory cytokines and chemokines secreted from ATMs promote inflammation, leading to localized and systemic insulin resistance and metabolic dysfunction. Nutrient metabolism and intracellular metabolic pathways also play critical roles in governing immune cell function and metabolic responses in nonimmune cells. Mitochondrial metabolism and the production of metabolites have been identified as crucial regulators of inflammatory signaling and macrophage phenotype.

[0082] One such metabolite is itaconate (ITA), derived from the tricarboxylic acid (TCA) cycle through the enzyme cis-aconitate decarboxylase- 1 (Acodl / Irgl). Acodl and ITA are among the highly upregulated genes and metabolites during pro -inflammatory macrophage activation. A recently discovered function of ITA and its isomers, mesaconate (MSA) and citraconate (CTA), is their differential activities for macrophage immunomodulation, where all three isomers inhibited glycolysis to a similar extent, altered amino acid metabolism, modulated cytokine / chemokine release and reduced interferon signaling, and oxidative stress.

[0083] ITA has exhibited anti-inflammatory and anti-oxidative effects in vitro, primarily attributed to multiple mechanisms such as succinate dehydrogenase (SDH) inhibition, induction of ATF3 and Nrf2, and inhibition of NLRP3 inflammasome activity. However, neither MSA nor CTA affected SDH activity or oxidative phosphorylation. Succinate (SCA), enhanced during inhibition of SDH by ITA) can activate AT thermogenesis. The approach used here is to improve the uptake and bioactivity of the natural metabolites through synthesis of metabolitebased biodegradable polyesters and subsequently into immunomodulatory nanoparticles (iNPs) or IMPRINT-NPs (Immuno Modulatory Particles for Reversing INflammation and phenotype Transformation), to assess their impact on obesity and other inflammatory disease outcomes.

[0084] There are conflicting reports regarding the ability of ITA to modulate macrophage polarization, suggesting its effects are context-dependent. In one study, ITA reduced M2 macrophage polarization through targeting Janus Kinase 1 (JAK1), yet in others, ITA induced M2 polarization in microglia or a mouse model of atherosclerosis through activation of Nuclear factor erythroid 2-related factor 2 (Nrf2). A recent study showed that daily oral administration of high dose ITA was effective as a prophylactic strategy to reduce the onset of obesity in mice that were transitioned to a high-fat diet, as ITA treatment increased both thermogenesis and fatty acid oxidation in AT. Despite these results, the frequent and high dosing requirements of ITA, likely due to poor cell membrane permeability, necessitates the development of an improved andsustained immunomodulatory approach to inhibit inflammation, functionally reprogram macrophages, induce fat browning, and reduce weight gain to tackle obesity.

[0085] To overcome the cell membrane permeability challenge associated with ITA, cell membrane-permeable derivatives of ITA, such as dimethyl itaconate (DMI) and 4-octyl itaconate (4-OI), have been developed and utilized to target immunometabolism and inflammation in diseases. DMI and 4-01 have been employed for immune modulation but do not recapitulate the effects of endogenous ITA and their untargeted delivery necessitates high doses that can result in toxicity due to poor solubility, requiring the use of organic solvents. Moreover, when administered in high doses, immune paralysis and off-target toxicity have been observed.

[0086] To solve this problem, the invention here relates to polymers synthesized from bioactive metabolites and formulation into nanoparticles (iNPs or IMPRINT-NPs). Without wishing to be bound by theory, this preparation is used to overcome these challenges, while avoiding the noted side effects of the medicament. Delivery of immunomodulatory reagents holds great promise for treating several immune- mediated conditions, including obesity. As an example, localized delivery of metabolites for immunomodulation presents a promising approach for the treatment of fat-related conditions, as it offers two key advantages: 1) it provides sustained modulation of the immune response, while minimizing off-target adverse effects on the overall immune system, and 2) it expands the range of immunomodulatory options by offering the ability to induce differential local and systemic effects to mitigate fat development at distance sites, thus overcoming limitations associated with diffusion-limited drug distribution.

[0087] In certain embodiments, the inventive NPs are useful alone in treatment of immune diseases and inflammatory diseases or infectious diseases, including obesity, allergic airway disease (AAD), and sepsis. In other embodiments, the iNP or IMPRINT-NPs can be developed for intravenous, oral, lung, intranasal, or subcutaneous delivery for the treatment of obesity and other inflammation-mediated diseases and conditions. Route of administration is not deemed to be a limitation of the IMPRINT-NP platform. In other embodiments, metabolite-based polymers or IMPRINT-NPs can be used to prepare or loaded in polymeric scaffolds or microneedles to modulate various aspects of the inflammatory response. In other embodiments, metabolite-based polymers can be formulated into particle sizes in the micrometer range (MPs). Moreover, IMPRINT-NPs can also be prepared to deliver disease-relevant antigens (amino acid based or nucleic acid-encoded) or small molecules or other biologies for immunomodulatory applicationslike vaccine development, cancer treatment, autoimmunity treatment, organ transplantation, wound healing, fibrosis, infectious disease treatment, and others.

[0088] IMPRINT-NPs were developed as a ‘cargo-free’ and multifaceted immunomodulatory approach. In this study, metabolite-based (p)-polymers were synthesized from ITA, MSA, and CTA were prepared and subsequently used to formulate NPs that are biodegradable and provide sustained release of the natural metabolites from the polymers through degradation. Other drugs are suitable for use with the invention, however. The data presented here shows that pITA-, pMSA-, and pCTA-based NPs (IMPRINT-NPs), for example, can mitigate proinflammatory cytokine secretions such as IL-6, MCP-1, IFN-0, and TNFa from bone marrow-derived macrophages (BMM0s) induced by lipopolysaccharide (LPS) stimulation. The data also show that treatment of BMM0S that have been unstimulated, Ml -activated (LPS stimulation), or M2- activated (IL-4 stimulation) with pITA- or pMSA-NPs resulted in significant enhancements in the surface expression of CD206, a phenotypic marker of M2-like macrophages, and reductions in markers of macrophage activation, including MHCII and CD86. Structural and mechanistic studies provided insight in mitochondrial rearrangement, supporting M2 phenotype induction. This enhancement in M2-phenotype (CD206 expression) was not observed in cells treated with 4-OI, DMI, or ITA employed as controls. These results showed that IMPRINT-NPs can switch or enhance the polarization of undifferentiated or pre-differentiated macrophages (Ml-to-M2 switch or MO- or M2-enhancement). Notably, the relative increase in CD206 was concentration- and composition-dependent for IMPRINT-NPs.

[0089] IMPRINT-NPs have been developed for the treatment of obesity. Prior to administering any treatments, mice were first fed a high fat diet (HFD) (HFD-induced obesity mouse model) for 16-20 weeks to render them obese. pITA-, pMSA-, pCTA-NP or control treatments were then delivered via three weekly subcutaneous injections and their body weight was tracked over 30 days. A significant decrease in body weight was observed for pITA-, pMSA-, and pCTA- IMPRINT-NP treated obese mice. In one study, PBS and PLGA-NP control groups continued to increase body weight, whereas pITA-NP treatment led to a significant reduction compared to controls. In another study, pCTA-NP treatment led to a slightly more effective reduction in body weight compared to pMSA-NPs.

[0090] Besides tracking body weight using the HFD-induced obesity model, the local and systemic immunomodulatory activity of pITA-NP were evaluated. The polymer molecularweight used for pTTA-NP formulation was essential for modulating CD206 surface marker expression in bone marrow-derived macrophages (BMM0), allowing the selection of pITAuigh- NP for further analysis. Co-culture studies evaluated the cross talk between NP treated Ml- macrophages and adipocytes supporting their use for obesity. Treatment of BMM0s with pITA- NPs led to direct phenotypic changes that influenced adipocyte behavior, supporting adipocyte browning — a key process in increasing energy expenditure.

[0091] Three weekly subcutaneous injections of pITA-NP into AT of HFD-induced obese mice significantly reduced weight gain, reduced WAT, mitigated systemic inflammation, increased anti-inflammatory cytokine secretions, and induced the conversion of WAT to BAT over 30 days (the end of the study). Quantitation of inguinal (ING), axillary (AXI), visceral (VIS), and liver weights also showed marked reductions for pITA-NP treated animals compared to controls. Overall, pITA-NP was identified as a highly effective immunomodulatory therapeutic approach to modulate macrophage polarization, inhibit systemic and AT inflammation, increase BAT, and reduce weight gain in obese mice. Cumulatively, our results support that IMPRINT-NPs (pITA-, pMSA-, pCTA-NPs) could be employed as novel immunomodulatory anti-obesity treatment by effectively altering macrophage phenotype, which subsequently induces browning in adipocytes and results in significant weight loss with potential to be applied to treat other inflammatory diseases with imbalanced macrophage polarization.

[0092] pITA-NP treatment affected cytokine secretions in vivo, aligning with observed gene expression changes in AT. pITA-NP-treated mice exhibited significantly reduced body weight without any noted toxicity, underscoring the therapeutic potential of this approach. Importantly, unlike conventional weight loss medications, which often come with significant side effects and limitations, pITA-NP and IMPRINT-NPs offer an immunometabolism-based approach that modulates immune and metabolic pathways to achieve weight loss. This strategy presents an innovative approach to obesity management, potentially offering a safer and more effective alternative to current pharmacological options. See FIG. 1 for an overview of a proposed immunomodulatory mechanism for pITA-NP treatment for obesity.

[0093] Moreover, the ability of various IMPRINT-NP formulations to improve survival outcomes using a lethal LPS-induced endotoxemia model of sterile inflammation was assessed. PLA-PEMA, pITA-NP, and pMSA-NP treatments all improved survival of mice treated with a lethal dose of LPS. PLA-PEMA NPs appeared to be most effective, followed by pITA-NP andpMSA-NP, which performed similarly. IMPRTNT-NPs also have the potential to treat bloodborne infections, skin infections, and promote wound healing, and other applications.

[0094] IMPRINT-NPs were incubated with Gram-positive and -negative bacteria cultured to assess colony forming units (CFU). pMSA- and pCTA-NPs were most effective at reducing bacterial growth, specifically E. coll, P. aeruginosa, and .S'. aureus. pITA-NP and PLA-PVA NPs also showed some effectiveness, but it was generally not as effective as pMSA- and pCTA-NPs.

[0095] With respect to inducing antigen (Ag)- specific immune tolerance, several studies encapsulating Ag to induce immune tolerance, require the co-delivery of immunomodulators such as IL- 10, TGF-[3, rapamycin, or dexamethasone. Specifically, one study demonstrated a need to co-encapsulate rapamycin, an immunosuppressant with OVA Ag to induce Tregs and significant reduction in Ag-specific responses. Our previous work systematically studied the role of Ag loading on Th2 tolerance induction where optimal Ag loadings that did not induce anaphylaxis and inhibited Th2 responses in vivo without co-administration of other immune modulators were identified.

[0096] The current invention of metabolite-based polymers and the formulated IMPRINT-NPs were able to overcome cell permeability limitations of their soluble surrogates. To overcome poor cell permeability of the ITA and MSA metabolites, the biodegradable polymers pITA and pMSA, when formulated into NPs, can provide a sustained and controlled delivery of ITA and MSA to cells through ester bond degradation. Cell permeable surrogates such as 4-OI and dimethyl itaconate (DMI) are effective at mitigating allergic airway inflammation at high and daily doses, whereas our preliminary findings indicated that a single intratracheal dose (lung delivery) of the pITA-NPs and pMSA-NPs can mitigate allergic inflammation (plasma IgE and Th2 cytokines in the lungs), which is more clinically desirable. Furthermore, 4-OI and DI do not inhibit SDH, therefore likely not providing the same cytoprotective properties as ITA.

[0097] Additional findings have demonstrated that pMSA-NPs can induce the highest levels of M2 macrophages when applied to undifferentiated M0 macrophages or Ml (LPS)- or M2 (IL-4)- differentiated macrophages. For example, bone marrow-derived macrophages stimulated with LPS for 24 hours to induce an Ml phenotype and treated with poly(lactic acid) (PLA)-NP, pITA- NP or pMSA-NP, for up to 24 hours. The pMSA-NPs showed a 4-fold increase in CD206, a marker for M2 macrophages, when compared to pITA-NPs. The versatility of IMPRINT-NPformulations to modulate macrophage polarization provides a platform to treat other immune- mcdiatcd diseases such as autoimmune and allergic diseases by incorporating antigenic cargoes.

[0098] 2. Definitions

[0099] Unless defined otherwise, all technical and scientific terms use herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials used are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration, and any other measurements, quantities, or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.

[0100] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the inclusion of any other item, element, step, or group of items, elements, or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element, or step modified by the article.

[0101] As used herein, the term “about” means plus or minus 20 percent of the recited value, so that, for example, “about 0.125” means 0.125 ±0.025, and “about 1.0” means 1.0 ±0.2. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in specific non-limiting examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing. Furthermore, unless otherwise clear from the context, a numerical value presented herein has an implied precision given by the least significant digit.Moreover, all ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein. For example, a range of "less than 10" can include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all sub-ranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 4.

[0102] As used herein, the term “nanoparticle” (NP) refers to a nanometer- sized particle with at least one dimension measuring between 1 to 1000 nm.

[0103] As used herein, the term “microparticle” (NP) refers to a micrometer-sized particle with at least one dimension measuring between 1-50 pm.

[0104] 3. Embodiments of the Invention

[0105] A. Introduction

[0106] Certain metabolites discussed herein have been shown to decrease both glycolysis and proinflammatory cytokines, while activating Nrf2 and its downstream anti-oxidant genes. The alteration of metabolism or other immune signalling processes caused by these metabolites can skew macrophages from an Ml -like to an M2-like phenotype. By polymerizing these metabolites to produce immunomodulating polymers and formulating them into nanoparticles (IMPRINT-NP), precise amounts of each metabolite can be delivered over time to macrophages and generate an anti-inflammatory response, skewing towards M2, thus promoting metabolic homeostasis.

[0107] This invention relates to inherently anti-inflammatory immunomodulatory nanoparticles (IMPRINT-NP) that inhibit proinflammatory responses generated by innate immune cells and can also be loaded with therapeutic cargoes for controlled release. Cargo-less (drug-free) poly(lactic acid) (PLA)-based iNPs inhibited lipopolysaccharide (LPS)-induced macrophage activation via a composition- and time-dependent reduction of NF-KB p65 and p38 MAPK activation.

[0108] Moreover, iNPs blocked surface interactions of pathogen-associated molecular patterns (PAMPs) with macrophages, without directly interacting with the PAMPs themselves. In vivo, iNPs when applied locally and as a prophylaxis, improved the survival of mice to greater than 70% in a lethal mouse model of endotoxemia. iNPs have also shown efficacy in a mouse model of polymicrobial sepsis.

[0109] The PLA iNP platform is expanded here to encompass ITA-, MSA-, and CTA-based polymers and IMPRINT-NPs to achieve efficient immunomodulation in in vitro and in vivo studies. Disease models described encompass LPS-induced endotoxemia, high-fat diet (HFD)- induced obesity, and allergic airway disease (AAD). Additionally, the inherent anti-bacterial properties of these various immunomodulatory nanoparticles have been studied, particularly with Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.

[0110] The data show that pITA-, pMSA-, and pCTA-based polymeric nanoparticles (IMPRINT-NPs) can suppress IL-6 and MCP-1 in a concentration dependent manner. Bone marrow-derived macrophages treated with pITA-, pMSA-, or pCTA-NPs under various conditions resulted in significant enhancement in CD206 expression, that IMPRINT-NPs could induce profound M2-like macrophage polarization, which was not observed in any controls that including soluble metabolites or the cell membrane permeable derivatives (DMI and 4-OI), highlighting the unique biological activity of IMPRINT-NPs to induce immune cell responses. When delivered subcutaneously to mice that have been fed a high fat diet (HFD) (HFD-obesity mouse model) for 16-20 weeks, a significant decrease in body weight was observed using pITA-, pMSA-, or pCTA-NPs (IMPRINT-NPs) administered to obese mice. Moreover, treatment of mice with PLA-PEMA NPs, pITA-NP, or pMSA-NP followed by a lethal challenge with LPS, led to dramatic improvements in survival. Additionally, a single intratracheal administration of pITA- or pMSA-NPs significantly reduced total IgE and Th2 cytokines in an OVA / Alum- induced AAD mouse model, outperforming PLGA-NPs and 4-OI.

[0111] Based on the results of the studies, the specification presents evidence for the broad immunomodulatory and therapeutic activity of pITA-, pMSA-, and pCTA-based IMPRINT-NPs without need for frequent dosing. Thus, the results from these studies are not applicable only towards the treatment of the applications described here for the compositions of the invention. These various IMPRINT-NPs are contemplated for use in encapsulating small molecules, peptides, proteins, antibodies, nucleic acids, and other biologically relevant cargoes for various therapeutic applications. For example, the described nanoparticles can be applied for polymicrobial sepsis, traumatic injury, immune tolerance induction (autoimmunity, allergy, allogeneic cell transplantation), rheumatoid arthritis, inflammatory bowel disease, spinal cord injury, fibrosis, cancer, and many others. The broad-therapeutic potential of these nanoparticles is anticipated to open several distinct, yet complementary areas of research for disease treatment.

[0112] Thus, developing a therapeutic strategy to modulate the inflammatory response, which functionally reprograms macrophaagc phenotypes towards M2-likc, represents a promising therapeutic approach to treat inflammatory and other immune-mediated conditions, including obesity.

[0113] B. Drug Compositions

[0114] Any drug composition can be used with the nanoparticles according to this invention. Although metabolite-based compounds are preferred, any drugs are suitable for use. For example, anticancer compositions, antimicrobial composition, statin compounds, antiinflammatory compounds, immunomodulating compounds, and the like are suitable. As specific examples, ITA, MSA, CTA and polymers thereof (pITA, pMSA, pCTA), are contemplated for use with the invention.

[0115] C. Polymers

[0116] Metabolite-based polymers were created for use with the invention. Specifically, ITA, MSA, and CTA were polymerized as described below. Preferred polymers for use in the invention are polymers of ITA, MSA, and CTA, however polymers comprised of monomers containing dicarboxylic acid moieties and the like also are contemplated for use with the invention. Further, during the polycondensation reaction, we have implemented the use of decanediol (CIO), an aliphatic diol, to link individual metabolites together to form the polymers. Other aliphatic diols or hydrophilic diols that have different lengths of carbon molecules or different side chain properties would also be useful. The use of aliphatic diols to create metabolite-based polymers is not required and other compounds containing diols could be used, whether water soluble or water-insoluble, bio-active, or the like. Metabolite-based polymers also can be arranged into various architectures, such as dendritic, hyperbranched polymeric scaffolds, hydrogels, or electrospun scaffolds. The various metabolites can be modified to enable other chemistries to be used for polymer synthesis, such as acrylate, bromination, or the like, to enable addition or RAFT polymerization techniques to be employed. Cell membrane permeable derivatives of itaconate have been developed (e.g. 4-OI and DMI). We also envision that modifications to MSA and CTA could also be applied to enhance their cell membrane permeability in the absence of polymerization.

[0117] D. Nanoparticles

[0118] Immunomodulatory NPs (iNP or IMPRINT-NPs) according to the invention can be drug-free and have a high biocompatibility and efficacy. They show inherent anti-inflammatory effects, which can enhance the treatment of inflammatory diseases, obesity, and allergy. They have tunable size and a special ability to control the release of immunomodulatory reagents through degradation, which can modulate macrophages and influence their activation state and polarization. pMSA- and pCTA- are two types of immunomodulatory polymers, which previously never have been synthesized or assessed for immunomodulation. The proposed IMPRINT-NPs facilitate the delivery of unmodified metabolites via endocytosis and phagocytosis to modulate macrophage responses. Due to the hydrophilicity of these metabolites, their internalization by macrophages is difficult and requires high doses for efficacy. Thus, IMPRINT-NPs can enhance the delivery of immunomodulatory metabolites through polymer synthesis and nanoparticle formation to reduce the effective dose and toxicity.

[0119] iNPs can enhance macrophage survival, prolong the immune system modulation, and provide long lasting immune responses. Simply treating macrophages with NPs can improve their survival. Thus, by increasing macrophage survival using iNPs, the immunomodulatory effects of these cells can be prolonged, surprisingly allowing for a more sustained and coordinated immune response.

[0120] IMPRINT-NPs can provide sustained release of metabolites controlled by degradation. IMPRINT-NPs provide the advantage of sustained drug release through their gradual degradation over time, facilitating a controlled and gradual release of metabolites embedded within the polyester backbone. The degradation rate of IMPRINT-NPs can be adjusted to achieve the desired release profile, enabling extended drug release. This controlled release property reduces the frequency of dosing required and helps maintain steady concentrations in the body. Additionally, IMPRINT-NPs have the potential to enhance their residence at the target site, thereby potentially amplifying their therapeutic effects.

[0121] iNP compositions can be produced and vary as a function of composition, surface chemistry, and particle size. As a control, commercially available PLA, with similar molecular weight as the synthesized polymers, can be used, yielding 10 polymer compositions. Two surfactants poly(vinyl alcohol) (PVA) and poly(ethylene-alt-maleic acid) (PEMA), for example, can be used to alter both surface chemistries (hydroxyl versus carboxyl) and zeta potential (neutral versus negative). iNP size (e.g., 400 nm and 100 nm) can be controlled using a singleemulsion-solvent evaporation methods or using a hydrodynamic flow-focusing microfluidics system. The formulations can be characterized for their physicochemical properties, such as particle size, zeta potential, and polydispersity index (PDI) via dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Particle degradation rates also can be assessed. iNPs can be resuspended in PBS and plasma and heated to 37°C. At designated timepoints, preferably spanning at least one month, samples can be collected for analysis. Analysis by GPC, LC- MS / MS, and scanning electron microscopy (SEM) are suitable.

[0122] In general, the nanoparticle size preferably is about 50 nm to about 2000 nm, more preferably about 200 nm to about 800 nm, and most preferably about 250 nm to about 500 nm in diameter. The Zeta potential of the nanoparticles preferably is about -10 to about -100, more preferably about -10 to about -60, and most preferably about -20 to about -50. The polydispersity index of the nanoparticles preferably is about 0.05 to about 0.45, more preferably about 0.05 to about 0.3, and most preferably about 0.05 to about 0.2.

[0123] E. Pharmaceutical Compositions

[0124] Microneedles, MNs, are a preferred method to provide a pharmaceutical composition of the inventive nanoparticles by subcutaneous injection. This method provides local, sustained, and uniform delivery of IMPRINT-NPs to the fat tissue. Using microneedles (MNs) as a delivery method to regulate the release of IMPRINT-NPs offers several advantages, including allowing the uniform distribution of IMPRINT-NPs in fat tissue and painless and easy application to the patient, making them highly suitable for immunomodulation. In the context of chronic inflammation, achieving appropriate immune modulation requires controlled, uniform distribution of the immune-active materials for treatment. With administration by regular injection, a significant proportion of immunized NPs can either remain at the site of immunization or are quickly cleared from the body before interacting with macrophages. Therefore, MNs can serve as a suitable platform for engineering the immune system and specifically modulating macrophages to achieve desired immune responses. Simple subcutaneous injection also may be used to administer the nanoparticles.

[0125] MNs offer a large surface area for delivery and are non-invasive. MNs offer numerous advantages as a non-invasive delivery method. Compared to traditional needles, they provide a larger surface area for efficient absorption of drugs or NPs. MNs also allow for targeted delivery to specific skin layers or localized regions of the body. Moreover, they are user-friendly andcarry a reduced risk of injury, making self-administration a viable option. This has the potential to improve healthcare accessibility, particularly in remote or resource-limited areas.

[0126] MNs arc excellent platforms for delivery of immune modulatory reagents. They can be administered quickly, efficiently, and with minimal tissue damage. MNs are single-dose, preparation- free, easy to administer, and have reduced size to simplify storage, transportation, and waste disposal. Additionally, MNs offer improved drug transport into tissues such as the fat, dose sparing effects, and thermostability. Importantly, passive delivery of drugs deep into the fat is a unique feature that enables the eradication of inflammatory macrophages and modulation of adipocytes. Since the therapeutic delivery strategy is to treat fat using immunomodulatory reagents to modulate macrophage polarization, sustained release of immune modulatory reagents can engineer local immune response at the fat site and modulate white fat. Unlike other polymer-based implants (e.g., microparticles and implants), MNs remain at the implantation site; thus, they can deliver drugs to the fat tissue uniformly.

[0127] Therefore, in a preferred embodiment, metabolite-based IMPRINT-NPs, which restore the M2 / M1 macrophage balance to treat obesity could be produced by synthesizing a set of polymers based on ITA, MSA, and CTA to formulate IMPRINT-NPs and subsequently loading them in MNs to achieve local sustained release and immunomodulation to treat obesity. These IMPRINT-NP-MNs: (1) efficiently load and deliver IMPRINT-NPs to functionally reprogram Ml macrophages that home to fat tissue, (2) release IMPRINT-NPs at a controlled rate to engineer the immune system and polarize macrophages to anti-inflammatory status, and (3) alter the white fat to brown fat ratio to treat obesity.

[0128] Controlled delivery of immune active materials is highly preferred in engineering MNs for immunotherapy to avoid complications. To this end, loading protocols that maintain IMPRINT-NPs bioactivity and yield controlled release profiles with high therapeutic efficacies with minimum local side effects are highly preferred. Additionally, the release profiles of IMPRINT-NPs can be adjusted by changing the methacrylate ratio of gelatin (GelMa) in the MN polymer.

[0129] Because the fabrication process may affect the bioactivity of IMPRINT-NPs, hydrogel MNs, or metabolite-based polymer MNs can be used. Hydrogels are highly desirable biomaterials for controlled release applications due to their ability to encapsulate and release bioactive molecules, such as antibodies, drugs, and immune active materials. Their water-swollen network provides an ideal environment for embedding and delivering these biologically active agents. MN patches with favorable mechanical properties can be achieved in this manner. The MN patches can be fabricated by a mold-based method. IMPRINT-NPs release profiles can be controlled by tuning the crosslinking degree of GelMA as well as the degree of methacrylation. Similarly, metabolite-based polymers can be used, instead of IMPRINT-NPs, to prepare MNs using a mold-based method. To test the IMPRINT-NPs release profiles of the MNs in vitro, MNs loaded with iNPs are immersed into 5 mL DPBS containing collagenase type II (2 U mL’1). Maintained at 37°C, 100 pL of the DPBS is sampled at predefined time points. After the measurements, each sample is returned to the solution for IMPRINT-NPs release analysis. HPLC, for example, can be used to quantitate total IMPRINT-NPs release. The activities of released IMPRINT-NPs before and after release are tested using the bone marrow derived macrophage (BMDM) assay.

[0130] In vitro degradation of MNs can be analyzed as well. For this, MN can be retrieved from the solution at pre-determined time points, and the wet weights recorded after blotting. The degradation ratio of MNs can be calculated as: (Wt / Wo)xlOO% (where Wt is residual wet weight at different time points and Wo is the initial wet weight). The concentration of GelMA (15% - 30%) and crosslinking time (0 - 5 min) preferably is optimized to obtain a drug release profile (MN degradation profile) ranging from 1 to 2 weeks.

[0131] Prior to use in a particular subject or group of subjects, the activity of platform can be assessed by treating BMDMs or ATMs with IMPRINT-NPs (as control) or IMPRINT-NP-MNs at different concentrations. The platform efficacy also can be evaluated in the absence of TLR agonists to investigate non-specific immune activation as a control. Cell survival, antiinflammatory responses, IMPRINT-NP-MNs effects on inflammatory and Nrf2-Keapl signaling, metabolic pathway analysis, and RNA-seq analysis of BMDMs and ATMs all preferably are performed. The phenotype of BMDM and ATMs for MHCII, CD40, CD80, CD86, and PD-L1 expression also preferably is performed using flow cytometry. Moreover, expression of arginase 1 and CD206 preferably is measured as described herein.

[0132] According to certain embodiments of the invention, pharmaceutical compositions are provided that incorporate the nanoparticles in a traditional pharmaceutical carrier or vehicle, which will depend on the anticipated route of administration. Routes of administration are determined by the person of skill according to convenience, the health and condition of thesubject to be treated, and the location and stage of the condition to be treated. A pharmaceutically acceptable carrier refers to any convenient compound or group of compounds that is not toxic and that does not destroy or significantly diminish the pharmacological activity of the therapeutic agent with which it is formulated. Such pharmaceutically acceptable carriers or vehicles encompass any of the standard pharmaceutically accepted solid, liquid, or gaseous carriers known in the art. Subcutaneous injection into the fat is a preferred route of administration, preferably using a microneedle technology. For lung delivery, IMPRINT-NPs can be combined with excipients to develop dry powder inhalers or produced using techniques such as spray drying.

[0133] For injection, the nanoparticles can be formulated in a liquid, such as an aqueous solution, suspension, or emulsion. Such a formulation preferably is sterile and is contained in a package such as a vial or a pre-loaded syringe. A preferred method of administering the nanoparticles of the invention is an array of microneedles which is administered as a topical patch on the skin of the subject.

[0134] For use in wound healing, the inventive polymers and nanoparticles can be incorporated into or comprise wound dressings, such as by electrospinning or any other method known in the art, or can be formulated for topical application in formulations such as transdermal patches, ointments, creams, gels, and the like. Oral compositions such as capsules or liquids also can be formulated by any of the methods known in the art.

[0135] F. Subjects

[0136] Subjects contemplated to benefit from the invention preferably are humans, but also can include any mammal, such as farm animals, companion animals, zoo animals, laboratory animals, and the like. The invention is contemplated to be used in treatment of any class of antiinflammatory conditions, including hyperproliferative diseases such as cancer or any inflammation-related or immune-mediated condition. Examples of these inflammatory conditions include metabolic disorders such as obesity, type 1 diabetes and type 2 diabetes; autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, celiac disease, lupus, scleroderma, Sjogrens syndrome; cardiovascular diseases such as hypertension and heart disease; gastrointestinal disorders such as inflammatory bowel disease, Crohn’s disease and ulcerative colitis; respiratory diseases such as chronic obstructive pulmonary disease, asthma, COVID- 19; neurodegenerative diseases; long CO VID syndrome or other viral conditions; inflammation dueto trauma (chemical or pollutant exposure, pathogens like bacteria, viruses, and fungi, radiation, or physical trauma), and the like. Therefore, a subject in need is any subject that suffers from a condition related to inflammation such as those listed above, is suspected of suffering from such a condition, or is at risk for developing such a condition.

[0137] H. Methods of Treatment

[0138] Biocompatible and bioresponsive MNs are technology platforms for sustained drug release with the potential to be key players in the transdermal delivery of therapeutics. The biodegradable MN patch of this invention for the sustained delivery of drugs using a polymer patch can be engineered to adjust delivery rates from the nanoparticles based on the degree of crosslinking in the polymer. GelMA also can be used as the base for engineering biodegradable MNs.

[0139] Administration of the drug compositions, including polymers and / or nanoparticles, can be administered one time or in multiple administrations over time. Administration may continue for a day, several days, weeks, months, years, or as a lifetime course of treatment. The polymers, the nanoparticles, or larger microparticles made of the polymers discussed herein can be administered by any route contemplated by the person of skill. Such methods include, but are not limited to injection into any body site (e.g., intravenous, intramuscular, subcutaneous, direct local injection into a site of disease or infection, injection into a lymph node, intraperitoneal, and the like), microneedle injection, or by other methods such as inhalation, lavage, wound covering, transdermal patch, or any other method known in the art.

[0140] 4. Examples

[0141] This invention is not limited to the particular processes, compounds, compositions, or methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, systems, compounds, compositions and materials are now described.

[0142] All publications mentioned herein, are incorporated by reference in their entirety; nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0143] Example 1 : General Methods and Materials,

[0144] A. Reagents

[0145] Acid-terminated 50:50 poly(lactic-co-glycolic acid) (PLGA; B6013-1) (~0.17 dL / g inherent viscosity in hexafluoro-2-propanol; approximately MW 4.2 kDa) and acid-terminated poly(lactic acid) (PLA; B6014-1) (0.15-0.25 dL / g inherent viscosity in hexafluoro-2-propanol) were purchased from Lactel Absorbable Polymers™. Poly(vinyl alcohol) (PVA), chloroform, dichloromethane, methanol, itaconic acid, mesaconic acid, citraconic acid, 1,10-decanediol, Tin (II) Chloride, 4-methoxyphenol (MEHQ), d-Chloroform, dimethylsulfoxide (DMSO), d-DMSO, 4-Octyl itaconate, dimethyl itaconate was purchased from MilliporeSigma™. All antibodies and chemicals were sourced from Thermo Fisher Scientific™, unless otherwise noted. Trypsin- EDTA, Dulbecco’s phosphate-buffered saline (DPBS), penicillin / streptomycin (Pen / Strep), and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Gibco™.

[0146] ELISA kits for IL-6, IL-ip, TNF-a, IL- 10, and IL-4 were sourced from BioLegend™. Mouse Triiodothyronine (T3) and TSH ELISA kits were obtained from LSBio™, while the Mouse C-Reactive Protein / CRP ELISA kit was purchased from R&D Systems™. Collagenase Type II, LipidTox, and Dispase II were obtained from Fisher Scientific™, and LPS was sourced from MilliporeSigma™. Luminex kits were purchased from Thermo Fisher Scientific and included murine IL-4, IL-5, IL-13, IL-10, IL-17A, and IFNy.

[0147] B. Mice and Mouse Models

[0148] Female C57BL / 6J (5-7 weeks old) mice were purchased from The lackson Laboratories™. 6-8 week old BALB / c mice were purchased from Charles River Laboratories, Wilmington, MA and housed in a facility under pathogen free conditions. All mice handling procedures were approved by the University of Maryland, Baltimore Institutional Animal Care and Use Committee (IACUC) or the Terasaki Institute IACUC.

[0149] The lethal LPS-induced Endotoxemia mouse model was used as a sterile model of sepsis. For this model, male C57BL / 6I (5-7 weeks) were injected interperitoneally with 2 mg of NPs 3 hours prior to intraperitoneal LPS administration at 20 mg / kg and monitored for 7 days. Modified Murine Sepsis Scoring (MSS), 20% > weight loss and death were used as endpoints.

[0150] For the high fat diet (HFD)-induced obesity mouse model, male C57BL / 6 mice (8-week old) were obtained from the Jackson Laboratory™ and were housed in a temperature-controlled room (22 ± 2°C) with a 12-hour light / dark cycle and free access to water. The mice wereseparated into non-HFD and HFD groups. Obesity was induced in the HFD group by feeding them HFD for 16-20 weeks, while the non-HFD group was fed a standard normal diet (Fisher Scientific™). Body weight was measured twice weekly, and once the HFD mice exhibited a 30- 35% increase in body weight compared to the non-HFD mice, the HFD mice were then grouped together based on their weight to achieve uniform weight distribution between treatment groups. The mice were given at least one week of adaptation before the experiments were conducted.

[0151] For some studies, all HFD-induced obese mice were divided into three groups: (1) PBS negative control (n=5); (2) PLGA-NP (n=5); and (3) pITAhigh-NP (n=5). Other studies included PBS negative control (n=5), pMSA-NP (n=5), pCTA-NP (n=5). All groups received injections of the respective treatment or control solution into both sides of the fat pads (inguinal (ING), visceral (VIS), and axillary (AXI)). At day 30, mice were euthanized using CO2 asphyxiation and the adipose tissues (ATs) with surrounding tissues and vital organs (heart, kidney, liver, spleen, and lung) were harvested to evaluate the potential systemic toxicity and efficacy of the treatments. ATs around the collected organs (kidney) were also collected to visualize the brown and white fat production near each tissue.

[0152] For metabolite-based NP treatments for allergic lung disease or allergic airway disease (AAD), female 6-8-week-old BALB / c mice (n=5 per group) were immunized intraperitoneally, on days 0 and day 7 with 10 pg of ovalbumin (OVA; grade V; MilliporeSigma™) in Alum (3 mg). Mice were intratracheally administered with 1 mg of PLGA-NP, pITA-NP, pMSA-NP, or 0.25 mg / kg (in 2% DMSO) of 4-octyl itaconate (4-OI) on day 14 relative to immunization prior to OVA aerosol challenge. The soluble control 4-OI was administered to the 4-OI group prior to OVA challenge for a total of 4 doses. OVA aerosol challenge occurred on day 15-17 with 10 mg / mL OVA for 20 minutes. Sample collection occurred on day +18. Control mice were immunized with PBS / Alum.

[0153] C. Isolation and Generation of Primary Bone Marrow-derived Macrophages (BMM0s).

[0154] C57BL / 6J (5-7 weeks old) mice were purchased from The Jackson Laboratories™ and housed in a facility at the University of Maryland, Baltimore, under pathogen free conditions. All mice handling procedures were approved by the University of Maryland, Baltimore Institutional Animal Care and Use Committee (IACUC). The femurs and tibias from 5-12 week old C57BL / 6J mice were isolated and flushed with BMM0 media (RPMI 1640 supplemented with L-glutamine (Life Technologies™), penicillin (100 units / mL), streptomycin (100 pg / mL),10% heat-inactivated fetal bovine serum (FBS) (VWR, Radnor, PA), and 20% L929 (ATCC, Manassas, VA) cell-conditioned media) using a 1 mL syringe and a 25-gaugc needle. Red blood cells were lysed using ACK lysis buffer (Quality Biological™). Cells were incubated in 5% CO2 at 37°C and media was changed on days 3, 6, and 8. Experiments were performed using day 8- 10 BMM0s.

[0155] D. Isolation of Mouse BAT Adipocytes.

[0156] ING, VIS, and AXI adipose tissues were dissected and processed to isolate adipocytes.The tissue was minced using a surgical blade and digested in a digestion buffer consisting of 10 mM CaCh, 3.5 mg / ml Dispase II, and 1 mg / mL collagenase II in IX DPBS for 60 minutes at 37 °C while shaking. EDTA (2 mM) was added to stop digestion, and the cells were then filtered using a 100 pm strainer (PluriSelect™) with in IX DPBS (10 mL) containing 3% BSA. Cells were kept in a Falcon™ tube for 1-2 hours and the suspended cells were subsequently transferred into a 5 ml Eppendorf™ tube using wide pipette tips. Finally, the cells were filtered through a 70 pm cell strainer, washed with 3-5 mL of PBS containing 3% BSA, and stained for further analysis by flow cytometry (Ze5, BioRad™) according to a previously published method.

[0157] E. Pathway Analysis; Western Blotting.

[0158] To evaluate the alterations in cell signaling induced by the nanoparticles, BMM0s were seeded at 1 x 106cells / well in sterile 6 well plates with and without LPS overnight. Cells were then treated with 30 pg / mL nanoparticles, or 125 pM of 4OI, for 3 hours then harvested.Samples were lysed using RIPA buffer with halt protease inhibitor, in combination with sonication. Samples were prepared by combining Lysates 50 / 50 with SDS Page running buffer and run on a 4-12% Tris Bis NuPage gel then immunoblotted using IRG1 and NF-K p65. Enhanced luminol-based chemiluminescence (ECL) was used for detection of the western blot.

[0159] F. Lung Hematoxylin and Eosin Histological Sectioning.

[0160] The lung was perfused with 5 mL of cold 10% buffered formalin and then placed in cassettes for further fixation for 24 hours. Samples were then paraffin embedded and sectioned into 5 pm slices. Hematoxylin and eosin (H&E) staining was used to characterize tissue architecture and immune cell mononuclear cell infiltration using standard procedures by the Pathology Biorepository Shared Services Core at the University of Maryland, Baltimore. The slides were imaged at a magnification of 10 times.

[0161] G. Assessment of NP effects on mitochondrial dynamics

[0162] To examine mitochondrial dynamics in bone marrow macrophages, Day 8 BMM0S were seeded at 5 x 104cclls / wcll in complete BMM0 media in sterile eight-well chamber slides incubated at 37°C and 5% CO2overnight to allow for cell adherence. The following day, media was replaced with fresh complete media supplemented with 100 ng / mL of LPS for 3 hours.These cells were then washed and treated with 30 g / mL of NPs for another 3 hours. The control group was treated with 125 pM of 4-OI in complete media for 3 hours. After 3 hours, the cells were washed twice with lx PBS to remove any extra particles and were fixed with fixation buffer (BioLegend™) per manufacturer’s instructions. Cells were then permeabilized using 0.1% Triton X-100 and blocked with 2% bovine serum albumin (Millipore Sigma™). The cells were then stained with rabbit TOM20 (#42406) (CST™) for 3 hours at room temperature. Cells were subsequently washed with lx PBS remove excess primary antibody and then stained with goat-anti rabbit IgG (H+L) Alexa™ Fluor Plus 488 antibody (# IC1051G) (Invitrogen™) for 1 hour. These cells were washed, and chambers were removed. Fluoroshield with DAPI (Millipore Sigma™) was used to coverslip and seal the slides, and placed in 4 °C overnight before imaging. Cells were imaged using Nikon Eclipse™ Ti-2 confocal microscope within 2 days.

[0163] H. Statistical Analysis

[0164] All statistical analyses were performed using GraphPad™ Prism software (version 10.0, GraphPad™ Software Inc.). Data are presented as mean ± standard error of the mean (SEM) unless otherwise stated. One-way and two-way, post-hoc Tukey ANOVA analyses determined the differences between the data. Statistical significance was indicated as follows: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001), and ns for non-significant differences.

[0165] Example 2: Metabolite-based Polymer Synthesis.

[0166] Metabolite-based polymers based on itaconic acid (pITA), mesaconic acid (pMSA) and citraconic acid (pCTA) were synthesized using a one-pot polycondensation reaction by reacting equal equivalences of commercially-available metabolites with 1,10-decanediol in the presence of a radical inhibitor (4-methoxyphenol (mequinol, MEHQ), 0.5 wt %) and a catalyst (SnCh, 10 mol %) under argon at 170°C with a dean stark distilling receiver attached to remove water. SnCh is not required, but provides improved yields.

[0167] The reaction was stopped at different timepoints, and the polymer was dissolved in 6 mL of chloroform and precipitated dropwise into 250 mL of ice-cold stirring methanol. The polymer was vacuumed filtered and dried under vacuum for 48 hours to remove any residual solvent.

[0168] Metabolite-based polymers can be synthesized via polycondensation reaction using diols of varying lengths and physicochemical properties and different immunomodulatory metabolites including itaconate, mesaconate, and citraconate. See the example polymer synthesis scheme and characterization of pITA-ClO (top), pMSA-ClO (middle), and pCTA-ClO (bottom) below.

[0169] The products formed were collected, dried, and characterized via proton nuclear magnetic resonance (1J f-NMR), Fourier-transform infrared spectroscopy (FT-IR) matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) and gel permeation chromatography (GPC) for structural conformation and molecular weight. See Table 1, below for data on GPC results.

[0170] Table 1. GPC Results.

[0171] Polymer samples for ’H-NMR were dissolved at 10 mg / mL in d-chloroform, while monomers were dissolved in DMSO-d6. Representative1H-NMR characterization of pITA-ClO demonstrated characteristic peaks associated with the pendant unsaturated carbonyl of ITA as well as the methylene hydrogens associated with the decanediol (CIO) indicating successful synthesis. Moreover, characteristic peaks related to pMSA-ClO and pCTA-ClO were also observed using ’H-NMR. A set of metabolite-based IMPRINT-NPs (pITA-NP, pMSA-NP, pCTA-NP, and PLA NP (as control)) were prepared with controlled sizes between 300-700 nm with negative zeta potentials between -20 to -30 mV.

[0172] Samples for molecular weight analysis via gel permeation chromatography (GPC) were dissolved at 5 mg / mL in chloroform overnight, filtered using a 0.2 pm filter, then injected on Styragel HR4E and HR3 columns in tandem, at a rate of 1 mL / min using a Waters™ Acquity HPLC system. Triple detection allowed for absolute molecular weight determination using a Malvern Panalytical™ OMNISEC detector. Analysis was performed using Malvern™ OMNISEC vl l.41 software.

[0173] Example 3: Nanoparticle Production and Formulation.

[0174] Nanoparticles were produced using oil-in-water (o / w) emulsion- solvent evaporation (SE) method with poly (vinyl alcohol) (PVA) or poly (ethylene-alt- maleic acid) (PEMA) as surfactants. 100 mg of pITA-ClO, pMSA-ClO, and pCTA-ClO were dissolved in chloroform at 50 mg / mL in a 20 mL scintillation vial (100 mg batch size). Any undissolved material was pelleted and removed by centrifugation. The polymer solution was transferred to a new 20 mL scintillation vial, then 10 mL of 1% PVA was slowly added to form two layers. The solution was sonicated using a Cole-Parmer™ 500 W Ultrasonic Homogenizer on ice using a 1 / 8* inch tip, for 30 seconds at 40% amplitude to make pITA-ClO, pMSA-ClO, pCTA-ClO PVA NPs. For PLA- PEMA or PLA-PVA NPs (as control), 100 mg of PLA was dissolved in dichloromethane at 50 mg / mL in a 30 mL beaker. 10 mL of 1% PEMA was added then sonicated at 100% amplitude a 1 / 4* for 30 seconds using the same homogenizer. The resulting emulsions were immediately poured into a beaker containing 80 mL 0.5% PVA (or 0.5% PEMA) and stirred overnight. Theparticle suspensions were filtered using a 40-micron cell strainer and subsequently washed three times using 40 mL of cold diFhO at 12,000 xg for 20 minutes.

[0175] The final NP pellet was resuspended and aliquoted into 2 mL screwcap microcentrifuge tubes with cryoprotectant (4% w / v sucrose, 3% w / v mannitol), frozen at -80°C, then lyophilized for 2 days prior to use. To generate Cy5.5-labeled NPs, Cy5.5-polymer conjugates were first synthesized, then formulated using 0.5% (w / w) conjugate into the various NP formulations. Nanoparticles were characterized via dynamic light scattering (DLS) (Malvern Panalytical™ Zetasizer ZSP) and nanoparticle tracking analysis (NTA) (Malvern Panalytical™ NanoSight NS300) to determine their size, polydispersity index (PDI) and zeta potential. The results of further characterization of IMPRINT-NPs are shown in Table 2, below. The values listed are the representative characteristics of the nanoparticles utilized for the experiments.

[0176] Table 2, IMPRINT-NP Characterization.

[0177] Additional data are presented in FIG. 3 A and FIG. 3B. These figures show data on the change in diameter and the change in zeta potential of pITA-NP and pMSA-NP, respectively, with time and temperature.

[0178] Example 4: Synthesis, Characterization, and Formulation of plTA-NPs.

[0179] A polycondensation reaction was used to synthesize polymers as above, and polymers were analyzed via gel permeation chromatography to determine polymer Mn, Mw and polydispersity index (PDI). pITA of three different molecular weights (low, med, high) were synthesized by bulk polymerization of itaconic acid and 1,10-decanediol in the presence of SnCk and MEHQ as a catalyst and radical inhibitor, respectively. The molecular weight of pITA was controlled by altering the synthesis time from 0.5 hour to 2 hour. The absolute molecular weight of pITA was determined using gel permeation chromatography with triple detection, concluding that pITA-ClO polymers had Mnvalues between 1 and 30 kDa with polydispersity indices less than 2.7. The monomers and pITA variants were further characterized using1H-NMR, which confirmed the polymer backbone structure, and the presence of the acrylate group associated with ITA. See Table 3, below.

[0180] Table 3. Polymer Characteristics.

[0181] Nanoparticles were formulated from PLGA and three types of pITA using a single emulsion- solvent evaporation technique. Particle diameter, polydispersity (PDI) and zeta potential were evaluated using dynamic light scattering (DLS) and determined that their size ranged from 250-650 nm with PDIs below 0.35, and zeta potentials near -20 mV. Results from nanoparticle tracking analysis are shown in FIG. 3. See Table 4, below for DLS characterization.

[0182] Table 4. Nanoparticle Characteristics.

[0183] Next, the degradation profile of PLGA-NP and pITAjiigh-NP was assessed by measuring the bulk mass decrease over one month at neutral pH (7.4) or slightly acidic pH (5.2) sodium acetate buffer to mimic biologically relevant environments. PLGA-NP degraded fully over 1 month at neutral pH and at 2 weeks at pH 5.2. No significant degradation was observed for pITAuigh-NPs under hydrolytic conditions. See FIG. 4.

[0184] Example 5 : Particle Uptake, Alterations of Mitochondrial Morphology, and Effects of pITA-NP treatment (molecular weight-, time-, and concentration-dependence) on Modulating Bone marrow-derived Macrophage Phenotypes.

[0185] We evaluated the ability of primary macrophages to take up nanoparticles using both confocal microscopy and flow cytometry; this ensured uptake of the nanoparticles. BMM0S were seeded at 5 x 104cells per well in an 8-well glass Millicell EZ chamber slide (MilliporeSigma™). To induce an Ml -like phenotype, cells were pre- treated with 100 ng / mL LPS overnight prior to NP treatment, MOs remained unstimulated. Cells were treated with 30 pg / mL of Cy5.5-labeled nanoparticles for 3 hours, washed twice with PBS, then fixed with fixation buffer. Fluoroshield with DAPI (MilliporeSigma™) was used to mount and stain the slides, a coverslip was immediately added. Once dried, slides were sealed and stored at 4°C until images were taken. A Nikon™ Eclipse Ti-2 confocal microscope was used to image slides.Confocal microscopy revealed that in LPS and non-LPS stimulated macrophages, both pITA and PLGA NPs are taken up by the cells at 3 hours. See FIG. 5 A.

[0186] For flow cytometry, particle uptake (pITA-NP-Cy5.5 and PLGA-NP-Cy5.5) was tracked over the course of 24 hours in M0 and Ml BMM0S. M0 macrophages were not exposed to any differentiation conditions prior to pITA-NP or PLGA-NP treatment. MI macrophages were generated by treating M0 macrophages with LPS overnight, followed by pITA-NP or PLGA-NP treatment. Cells were collected and stained in accordance with BioLegend™ protocols for flow cytometry. Data were collected using Cytek Aurora™ flow cytometer and analysis was performed using FCS Express™ 7 (De Novo™ Software). Staining with Live / Dead GreenViability / Cytotoxicity (Thermo Fisher Scientific™) was done prior to FcR blocking with anti- CD16 / 32 antibody. See FIG. 5B.

[0187] To assess how treatment with various IMPRINT-NPs influenced mitochondria morphology, we analyzed mitochondria dynamics using Day 8 bone marrow-derived macrophages (BMM0s) polarized to the Ml phenotype using LPS. Cells were treated with pITAnigh-NP, PLGA-NP, 4-octyl-itaconate (4-OI), untreated (NT; MO), and LPS alone (Ml) as a control. The mitochondria were visualized using TOM20 staining and confocal microscopy. Consistent with previous findings, NT (MO) macrophages displayed short mitochondrial lengths, while LPS-treated Ml macrophages exhibited an elongated mitochondrial network.Interestingly, 4-01 treatment also resulted in an elongated mitochondrial morphology, similar to the LPS-treated group, which was unexpected. PLGA-NP treatment also led to a long mitochondrial network. Notably, pITAnig -NP-treated cells displayed short mitochondrial lengths, closely resembling the morphology of untreated MO and M2 macrophages. These findings suggest that pro-inflammatory Ml macrophages are associated with elongated mitochondria, while resting and M2-like macrophages maintain a shorter mitochondrial structure consistent with their phenotype. This indicates that pITAnigh-NP may promote a less inflammatory macrophage state by influencing mitochondria morphology. See FIG. 6.

[0188] MO macrophages were not exposed to any differentiation conditions prior to pITA-NP treatment. Ml macrophages were generated by treating MO macrophages with LPS overnight, followed by pITA-NP treatment. M2 macrophages were generated by treating MO macrophages with IL-4 overnight, followed by pITA-NP treatment. For these tests, the particle concentration was 30 pg / mL. Results are shown in FIG. 7.

[0189] MO macrophages were seeded in a 24-well plate at a density of 0.2 x 106cells / well, stimulated overnight to induce MO, Ml and M2 macrophages, and then treated with 30 pg / mL of pITAiow-, pITAmed-, or pITAhigh-NPs for 24 hours, in the presence of the respective macrophages stimulant. Flow cytometry was used to evaluate how pITA-NP treatment altered key macrophage polarization markers to identify the extent of NP induced changes, including MHCII, CD86, CD80, and CD206 exposure to these conditions.

[0190] For M0 macrophages, pITAnigh-NP reduced MHCII expression in a molecular weightdependent manner. CD80 expression was most significantly reduced with pITA ow-NP, with levels increasing as molecular weight increased. CD206 expression, used as an M2 polarizationmarker, was significantly elevated compared to the untreated PBS control, showing a 300% increase with pITAuigh-NP.

[0191] LPS, an inducer of Ml macrophage polarization, was used to evaluate the potential of pITA-NPs to reverse pre-established inflammatory responses. pITAuigh-NP treatment slightly reduced MHCII, CD86, and CD80 expression compared to the LPS-treated control. Notably, CD206 expression reached its highest levels with pITAMed- and pITAnigh-NPs. These results indicate that pITA-NPs can effectively dampen pre-established macrophage activation and repolarize Ml inflammatory macrophages towards an anti-inflammatory, M2-like phenotype.

[0192] We assessed the effects of pITA-NP on pre-established M2-polarized macrophages, where IL-4 was used to induce the M2 phenotype. Unlike the MO and Ml conditions, pITA-NP treatment of M2 macrophages led to a significant increase in MHCII expression, along with reductions in CD86 and CD80.

[0193] Consistent with the M0 and Ml conditions, pITA-NP treatment resulted in a significant enhancement in CD206 expression. Taken together, these results demonstrated the most effective pITA-NP capable of reducing macrophage activation and inducing anti-inflammatory M2 macrophage polarization was pITAuigh-NP.

[0194] A similar set of studies were performed using pITAhigh-NP, 4-octyl itaconate (4-OI), dimethyl itaconate (DMI), and itaconate (ITA) metabolite to compare how the various cell membrane-permeable itaconate derivatives (4-OI and DMI) or soluble itaconate affected MO and pre-established Ml -macrophage polarization. LPS (Ml macrophage control) and IL-4 (M2 macrophage control) were included as additional comparators. The results are shown in FIG. 8 and FIG. 9.

[0195] For M0 macrophages, pITAuigh-NP reduced MHCII and CD80 expression, while significantly increasing CD206 expression, used as an M2 polarization marker. Notably, treatment with 4-OI, DMI, or ITA alone did not induce any alterations in phenotype. See FIG. 8.

[0196] For Ml macrophages, pITAuigh-NP slightly increased MHCII compared to the LPS control, while significantly increasing CD206 expression, used as an M2 polarization marker. IL- 4 treatment, employed as a control for M2 macrophage polarization only slightly increased CD206 expression. DMI slightly reduced CD80 expression compared to LPS control. Once more, treatment with 4-OI, DMI, or ITA alone did not induce any alterations in phenotype.These results highlighted that pITA-NP treatment could modulate macrophage phenotypes in a distinct manner opposed to 4-OI, DMI, and ITA alone. Sec FIG. 9.

[0197] Another set of studies were performed using MO and Ml macrophages to assess the time-dependent alterations in BMM0 polarization at 3, 8 and 24 hours following pITAuigh-NP and PLGA-NP treatment (30 .g / mL concentration). Interestingly, when evaluating the particles ability to alter surface marker expression, the particles did so in a composition and time dependent manner. As expected, when cells were stimulated with LPS there is an increase in CD86 and CD80 co-stimulatory marker expression, and when treated with particles, there was little effect on expression (see FIG. 10A and FIG. 10B). This trend remained the same over time. On the contrary, cells treated with pITAuigh-NPs saw a drastic increase in CD206 expression, more so than that of the PLGA-NPs. As time increased, the more expression of CD206 is seen in both LPS and non-LPS stimulated cells (FIG. IOC). CD206 being a marker for M2 polarization, this could indicate a phenotypic switch.

[0198] After 3 hours of treatment, the histograms in FIG. 11 show live, F4 / 80+and CD1 lb+cell surface molecule expression with the red line histograms representing the treatment groups while the grey filled histograms represent the untreated BMM0S.

[0199] In further testing, Ml macrophages displayed altered surface molecule expression using flow cytometry when treated with higher concentrations of pITAuigh-NPs that showed significant increases in CD206, as well as concentration-dependent decreases in MHCII, CD86, and CD80, something not seen with our control NPs or with the soluble itaconate control. See FIG. 12. Statistical differences were determined by one-way ANOVA with Tukey’s multiple comparisons test. Within data subsets, no significant difference was observed between conditions labeled with matching letters (p>0.05). Error bars indicate SD.

[0200] Example 6: IMPRINT-NPs Display Differential Anti-Inflammatory Properties for Cytokine Modulation.

[0201] The IMPRINT-NP polymer composition- and concentration-dependent effects on toxicity were assessed, as well as their ability to inhibit proinflammatory cytokine secretions from BMDMs treated with LPS (TLR4 agonist) compared to 4-octyl itaconate (4-OI), a cell membrane permeable itaconate derivative.

[0202] We assessed how various IMPRINT-NP formulations affected viability and the secretion of LPS-induced cytokines. Using ELISA, we measured IL-6 as a proinflammatory cytokineassociated with activated (inflammatory) macrophages and monocyte chemoattractant protein (MCP-1) was measured as it is involved in ATM recruitment to adipose tissue. Sec FIG. 13. pITA-NP did not significantly reduce cytokine secretions at any concentration tested. pCTA-NP, pMSA-NP, and PLA-NPs were similarly effective at reducing IL-6 and MCP-1 secretions, however they were less effective than 4-OI control. At the highest concentration tested, we observed a similar viability (-75%) compared to LPS (93%) and 4-01 (77%) controls.

[0203] Example 7: ‘Cargo-free’ pMSA-NP are Highly Effective at Inducing CD206 Expression Compared to pITA-NP and PLA-NP to Skew Macrophage Polarization towards an Antiinflammatory M2-like Phenotype.

[0204] The molecular weight- and time-dependent effects of pMSA-NPs on macrophage polarization and activation were assessed under unstimulated (M0) or pre-established Ml- and M2-polarization conditions. MO macrophages were not exposed to any differentiation conditions prior to pMSA-NP treatment. Ml macrophages were generated by treating MO macrophages with LPS overnight, followed by pMSA-NP treatment. M2 macrophages were generated by treating MO macrophages with IL-4 overnight, followed by pMSA-NP treatment. For these tests, the particle concentration was 30 pg / mL and pITAuigh-NP and PLA-NPs were also employed as controls. Flow cytometry was used to evaluate how pMSA-NP treatment altered key macrophage polarization markers to identify the extent of NP induced changes, including CD206, CD80, CD86, MHCII. See FIG. 14, FIG. 15, and FIG. 16.

[0205] For MO macrophages, pMSA ow-NP significantly increased CD206 expression, used as an M2 polarization marker. CD80 expression increased as molecular weight of pMSA increased. CD86 was unmodified. MHCII expression reduced as a function of pMSA molecular’ weight. See FIG. 14.

[0206] For Ml macrophages, pMSAuow-NP treatment significantly increased CD206 expression, used as an M2 polarization marker. CD80 expression was reduced with pMSALow- NP and increased to the same level as LPS as molecular weight of pMSA increased. CD86 was unmodified. MHCII expression was reduced for all pMSA-NP groups tested independent of molecular- weight. These results indicate that pMSA-NPs can effectively dampen pre-established macrophage activation and repolarize Ml inflammatory macrophages towards an antiinflammatory, M2-like phenotype. Importantly, pMSAi.ow-NP induced multiple fold change higher expression of CD206 compared to the control NPs employed. See FIG. 15.

[0207] We assessed the effects of pMSA-NP on pre-established M2-polarized macrophages, where IL-4 was used to induce the M2 phenotype. Similar to the MO and Ml conditions, pMSA- NP treatment of M2 macrophages led to a significant increase in CD206 compared to controls. CD80 expression was reduced for pMSALow-NP and subsequently increased as molecular weight of pMSA increased. CD86 was unmodified. Interestingly, MHCII expression was significantly increased for pMSArow-NP, which was not observed for the other pMSA-NP formulations tested. Taken together, these results demonstrated the most effective pMSA-NP capable of reducing macrophage activation and inducing anti-inflammatory M2 macrophage polarization was pMSArow-NP. See FIG. 16.

[0208] Example 8: IMPRINT-NP are Non-toxic when Administered to Mice.

[0209] To evaluate any potential toxicity of IMPRINT-NPs in animals, we administered IMPRINT-NPs (pITA-, pMSA-, and pCTA-NP) intravenously (2 mg / dose) into C57BL / 6 mice, and after 24 hours, blood was collected for analysis. No significant alterations in aspartate aminotransferase (AST), alanine aminotransferase (ALT), or creatinine kinase were measured compared to the control. See FIG. 17.

[0210] Example 9. Intratracheal Administration of IMPRINT-NPs Reduces Plasma IgE, Th2 Cytokines in the Lung, and Eosinophil and Macrophage Infiltration Associated with AA.

[0211] Female BALB / c mice were sensitized by OVA / Alum immunization (dO, d7) and mice were administered a single dose of IMPRINT-NPs (pITAnigh-NP or pMS ALOW-NP) intratracheally (i.t) in PBS (1 mg / dose) on d!4. The control treatment, 4-OI was administered daily (in diluted dimethyl sulfoxide) i.t. to the control group on dl4-17 (prior to challenge). Mice were challenged with aerosolized OVA (10 mg / mL) for 20 min for three consecutive days. Bronchoalveolar lavage fluid (BALF) and plasma were collected on dl8. PLGA-NPs were also used as a control as it has been used by our lab as a carrier for antigens to treat allergic airway disease. See FIG. 18 A.

[0212] A single dose of pITAnigh-NP or pMSAtow-NP significantly decreased total IgE and Th2- associated cytokines (IL-4, IL-5, and IL-13, as well as IFNy); however, PLGA-NPs had no effect on IgE and was less effective for Th2 cytokines. While 4-OI did significantly reduce IgE and cytokines, it was provided as a daily dose prior to aerosol challenge and required the use of an organic solvent for dissolution. The ability for IMPRINT-NPs to reduce total IgE and effectively mitigate Th2 cytokines more effectively than 4-OI and PLGA-NP supports their furtherdevelopment as an Ag-specific NP immunotherapy for AAD and other inflammatory diseases. While this reduction in total IgE and Th2 associated cytokines was not antigcn-spccific, these results support the development of IMPRINT-NPs as a platform for antigen-specific treatment for autoimmune and airway inflammatory diseases. See FIG. 18B and FIG. 18C through FIG. 18F.

[0213] Plasma, BALF and blood were collected on day 18 and processed using ELISA for total IgE and MAGPIX Luminex™ bead-based multiplex ELISA (Thermo Fisher Scientific™) diluted 2-fold and measured for various cytokines. For collection of BALF, lungs were immediately lavaged after euthanasia with a trachea cannula with 0.8 mL of bronchoalveolar lavage fluid [BALF; 1 mM EDTA and 10% (vol / vol) FBS in PBS]. The collected BALF was centrifuged at 500 x g for 3 minutes at 4°C. The supernatant was collected and stored for further analysis. The Luminex data were analyzed using the Luminex™ xPONENT software (Millipore™) per the manufacturer’s instructions. The panel included murine IL-4, IL-5, IL-13, and IFNy.

[0214] The BALF collected from mice used in experiments described in FIG. 18, above, were centrifuged to pellet the cells collected during the lavage. The whole cells from the BALF were analyzed and resuspended in 0.5 mL of lx PBS, and the total number of cells was counted using a hemocytometer. The samples were subjected to cytocentrifugation (Cytospin 3, Thermo™) onto slides, and the slides were stained using the Diff-Quick™ (Medion Diagnostics™) staining solution to identify and count the immune cells, including macrophages, neutrophils, eosinophils, and basophils. The slides were observed using a Revolve™ light microscope (ECHO) at a magnification of 10 times. These cells were resuspended in PBS and counted for differential staining analysis to determine the cell types within the BALF. The differential staining results are shown in FIG. 19. The PBS group was shown to have minimal macrophages as expected (see FIG. 19A), while the no treatment group showed an increase in macrophages and eosinophils (see FIG. 19B). The cell permeable itaconate derivative, 4-OI (FIG 19C) and PLGA-NPs (FIG. 26D) did not reduce macrophage and eosinophil infiltration as much as pITA- NP (FIG. 19E) and pMSA-NP (FIG. 19F). The average macrophage and eosinophil counts are shown in FIG. 20.

[0215] Example 10: Effects of pITAuigh-NP on Macrophages and 3T3-L1 Adipocyte Cells.

[0216] NPs ability to alter adipocyte differentiation through macrophage crosstalk was investigated using 3T3-L1 cells (ATCC, VA, USA) cultured in a transwcll plate with M0s. Briefly, 3T3-L1 cells were cultured at 300,000 cells per well in 12-well plates and differentiated into mature adipocytes using a differentiation cocktail consisting of insulin (10 pg / ml, Sigma1M), dexamethasone (1 pM, Sigma™), and 3-isobutyl-l -methyl xanthine (0.5 mM, Sigma™) or any other known method in the art. LPS-activated macrophages were seeded on top of a 6 pm pore size cell membrane insert, which was then placed over 3T3-L1 cells. After 24 hours of coculture, 20 pg / mL of PLGA- or pITAhigh-NPs were added directly to the macrophages on the membrane inserts to study the impact of macrophage polarization on the differentiation of 3T3- L1 cells into mature adipocytes. The co-cultures were maintained for 24, 48, and 72 hours. At these time points, adipocyte differentiation was assessed by quantifying lipid droplet (LD) accumulation inside the cells using LipidTox™ (Fisher Scientific™) and Phalloidin (Invitrogen™) staining kits. The total area, Feret’s Diameter (FD), and Integrated Optical Density (IOD) were quantified using ImageJ™ 1.33 software.

[0217] To explore the effects of pITAnigh-NPs on indirectly altering adipocyte phenotype, we examined the ability of pITAnigh-NP treated Ml polarized macrophages to alter fully differentiated 3T3-L1 white adipocytes in a transwell plate over 72 hours. See FIG. 21A. LipidTox and Phalloidin was used to visualize lipid droplet accumulation overtime. See FIG. 21B. Additionally, the total area, integrated optical density (IOD) and Fret’s diameter was calculated. See FIG. 21C through FIG. 21E.

[0218] Little difference was observed between the no treatment control group and the PLGA-NP treat groups, however significant differences were seen with macrophages treated with pITAnigh- NP. Decreases in total area, IOD, and Fret’s diameter suggest a phenotypic switch of the 3T3-L1 cells from a WAT to BAT, as BAT has smaller lipid droplets. This further confirms the ability of macrophages to directly impact adipose phenotype and corroborates the ability of pITAnigh- NP to induce browning.

[0219] Example 11: Efficacy Study of IMPRINT-NPs using a Mouse Model of HFD-induced Obesity.

[0220] To determine the efficacy of iNP (pITAnigh-NPs, pMSALow-NPs, and PCTALOW-NPS), the high fat diet (HFD)-induced obesity mouse model was employed. Briefly, male C57BL / 6 mice (8-weeks old) were obtained from the Jackson Laboratory™ and were housed in a temperature-controlled room (22 ± 2°C) with a 12-hour light / dark cycle and free access to water. The mice were separated into non-HFD and HFD groups. Obesity was induced in the HFD group by feeding them HFD for 16-20 weeks. Body weight was measured twice weekly throughout the course of the study, and once the HFD mice exhibited a 30-35% increase in body weight compared to the non-HFD mice, the HFD mice were then grouped together based on their weight to achieve uniform weight distribution between treatment groups. The mice were given at least one week of adaptation before the experiments were conducted. All mice were continually allowed access to HFD chow throughout the entire study time period. See FIG. 22A for the general treatment scheme for IMPRINT-NPs.

[0221] For some studies, HFD-induced obese mice were divided into three groups: (1) PBS negative control (n=5); (2) PLGA-NP (n=5); and (3) pITAhigh-NP (n=5). See results for weight change in FIG. 22B. Other studies included PBS negative control (n=5), pMSA-NP (n=5), pCTA-NP (n=5). See results for weight change in FIG. 23. All groups received injections of 100 |lL total of treatment (20 mg / mL IMPRINT-NPs in PBS) or control, split into 6 smaller volume injections into both sides of the fat pads (inguinal (ING), visceral (VIS), and axillary (AXI)). Animals were dosed on days 0, 7, and 14. Six milligrams total were delivered over the course of the study. At day 30, mice were euthanized and the plasma and adipose tissues (ATs) with surrounding tissues and vital organs (heart, kidney, liver, spleen, and lung) were harvested to evaluate the potential systemic toxicity and efficacy of the treatments. ATs around the collected organs (kidney) were also collected to visualize the brown and white fat production near each tissue. Significant reductions in weight, as well as various fat tissues were observed for IMPRINT-NP (pITAnigh-NP, pMSAtow-NP, and pCTALow-NP) treated mice.

[0222] Further analysis was performed on samples collected from the study described in FIG. 22B. The sizes and weights of the ING, AXI, VIS fat pads, as well as the liver, all significantly decreased in our treatment mice. See FIG. 24. Representative histological samples depict the shrinkage of adipocytes within the liver samples, further denoted by the color change. See FIG. 25.

[0223] At the end of the in vivo study from FIG. 22B, blood was collected from mice, and serum was separated using BD Microtainer Tubes with Yellow Caps containing Serum Separator Gel, following the manufacturer's protocol. The collected serum samples were stored at -20°C until further analysis. ELISA assays were performed according to the manufacturer'sinstructions. Mouse ELISA kits for IL-6, IL-2, IL- 10, TNF-a, IL- 10, and IL-4 were purchased from BioLcgcnd™, while ELISA kits for TSH, T3, and C-rcactivc protein (CRP) were obtained from MyBioSource™. All assays were conducted following the specific procedures provided by the respective manufacturers. Following cytokine analysis, it was found that pITAuigh-NP was able to significantly decrease pro-inflammatory cytokines IL-6, IL- 10, IL-2, and TNF-a, while significantly increasing levels of anti-inflammatory cytokines, IL-4 and IL- 10. See FIG. 26. To further corroborate the results, serum C-reactive protein (CRP) levels, an inflammatory protein and common marker for inflammation, were evaluated and found to be significantly decreased. Thyroid function was also assessed, as it is commonly associated with obesity and plays a key role in metabolism; to account for function, both the thyroid-stimulating hormone (TSH) and triiodothyronine (T3) levels were evaluated. It was found that in pITAuigh-NP treated mice, the TSH levels decreased, while the T3 levels increased, suggesting that pITAuigh-NP impacts thyroid function. See FIG. 26.

[0224] To further evaluate the impact of particle treatment, the adipose tissue was sectioned and stained (H & E). Decreases in the size of adipocytes were observed in both the visceral fat (FIG. 27 A) and in the fat surrounding the kidneys (FIG. 27B), as well as within tissues themselves as depicted in FIG. 28. The calculated total area, Feret’s diameter, and the IOD quantitatively depicts the size reduction and lipid content reduction of adipocytes within the visceral adipose tissue. See results in FIG. 29. The ING, AXI and VIS fat pads were then processed to determine the phenotype of the adipocytes using UCP1 expression as a marker for BAT. Flow cytometry revealed that there was a significant increase in UCP1 expression in all fat pads that had been treated with pITAuigh-NPs, when compared to HFD and PLGA NP controls, indicating that pITAuigh-NP treatment induces browning within adipose tissue. See FIG. 30.

[0225] Lastly, to directly reveal the molecular changes that occurred in adipose tissues and the liver as a result of PLGA-NP or pITAjiigh-NP treatment, gene expression was investigated. Total RNA was extracted from liver, AXI, ING, and VIS ATs using Quick-RNA Miniprep Kits (Thomas Scientific™), according to the manufacturer's protocol. RNA quality and quantity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific™) and Agilent™ 2100 Bioanalyzer (Agilent Technologies™). Only samples with RNA integrity number (RIN) > 8 were used for further analysis.

[0226] cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™) following the manufacturer's protocol. Next, 1 pg of mRNA was reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific™) in a total volume of 20 pL. Quantitative real-time polymerase chain reaction (qRT- PCR) was performed with Fast SYBR Green Master Mix (Thermo Fisher Scientific™) on the Thermal Cycler CFX384TM Real-Time PCR System. The cDNA amplification conditions were 95 °C for 20 seconds followed by 45 cycles at 95 °C for 3 seconds and 60 °C for 30 seconds.The quantitative reactions were replicated five times. Relative target gene expression levels were measured using 2'AACtmethod and GADPH was used to normalize the expression data as an internal control gene. Hierarchical clustering of gene expression was performed to summarize the collected data. See FIG. 31. The primer sequences used for PCR amplification are listed in Table 5, below.

[0227] Table 5. Primer Sequences for qRT-PCR.

[0228] Looking specifically at genes that are involved in thermogenesis, a characteristic of brown adipocytes, we saw an upregulation in UCP1, Prdml6, Elovl3 and Cidea, as well as PPARyl which plays important role in adipogenesis. Prdml6 activates genes involved in thermogenesis, particularly Vcpl. It does this through its interactions with other transcriptional coactivators like PPARy and PGC-la. These interactions enable Prdml6 to stimulate the transcription of mitochondrial and thermogenic genes, enhancing energy expenditure. By generating heat and metabolizing fatty acids, Ucpl and Elovl3 work together to enhance energyexpenditure in BAT. Cidea enhances lipid storage in adipocytes, contributing to fat accumulation, particularly in the context of obesity, while modulating the uncoupling action of Ucpl.

[0229] Genes that are involved in adipocyte inflammation were also assessed, providing mixed results. In obesity, the balance between pro-inflammatory (aP2, Cyclophilin and Adipsin) and anti-inflammatory (Adiponectin) factors is disrupted, leading to a chronic inflammatory state that promotes insulin resistance, metabolic dysfunction, and further fat accumulation. Here, we found that pITA-NP treatment significantly increases Adiponectin in most tissue collections, which serves as an anti-inflammatory and insulin sensitizing hormone that is typically downregulated in obesity; reduced levels lead to greater insulin resistance and systemic inflammation.

[0230] On the contrary, both aP2 and Cyclophilin are increased, and they both disrupt adiponectin signaling. Adpisin, a gene typically used to identify white adipose tissue and is also known as compliment factor D in the alternative compliment activation pathway, is decreased in pITA treated samples. This suggests that systemic inflammation is decreasing.

[0231] Further assessing the effects of pITA-NPs on downstream thyroid function, Dio2 expression was evaluated. Dio2 encodes for D2, the enzyme that converts thyroxine (T4) to the active hormone triiodothyronine (T3). T3 plays a significant role in regulating metabolism, including thermogenesis in brown fat. This gene, however, was significantly decreased in all collected fat pads, but significantly increased in the liver. Interestingly, PGC-la, a protein that regulates energy metabolism and homeostasis, expression was increased despite needing the enhancement of thyroid hormone signaling induced by D2.

[0232] Principal component analysis (PCA) of Ctrl (obese control), PLGA-NP, and pITA-NP treatments revealed distinct clustering of pITAuigh-NP from the other groups. See FIG. 32.

[0233] Specifically evaluating genes associated with fat browning / thermogenesis and insulin sensitivity showed that they are significantly enhanced after pITAuigh-NP treatment. Ucpl (FIG. 33A) and Adiponectin (FIG. 33B) expression is significantly upregulated in multiple tissues.

[0234] Example 12: Efficacy of IMPRINT-NPs to Treat LPS-induced Endotoxemia.

[0235] The survival benefits of PLA-PEMA-NP, pITAuigh-NP, and pMSALoW-NPs were assessed using a lethal LPS-induced endotoxemia mouse model. Male C57BL / 6 mice (5-7 weeks) were administered various NPs (2 mg single dose) via intraperitoneal injection 3 hoursbefore 20 mg / kg intraperitoneal LPS injection and the survival was tracked over 7 days. As a control, mice subjected to LPS only were pretreated with saline. 40% of pMSA-NP-trcatcd mice survived following LPS challenge, while 60% of PLA-PEMA-NP and 50% pITA-NP- treated mice survived, compared to the control LPS-treated mice (20% survival). Overall, all NP formulations improved survival, however, pITA-NP and PLA-PEMA-NP induced the greatest improvement in survival. See FIG. 34.

[0236] Example 13: Antibacterial Activity of Soluble Metabolites and IMPRINT-NPs.

[0237] To assess the inherent antibacterial activity of IMPRINT-NPs, we employed a streak plate assay using three different types of bacteria strains. Two bacteria strains were Gramnegative (Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa)) and one was Gram-positive (Staphylococcus aureus (S. aureus)) . For E. coli, the UPEC strain was employed. For P. aeruginosa, the PAO1 strain was employed. For S. aureus, the N2 strain was employed. In these experiments, the effects of the individual metabolites, itaconate (ITA), mesaconate (MSA), and citraconate (CTA) were first tested by measuring their effects on colony forming units (CFUs) followed by their effects on bacterial growth using time-dependent growth curves. See FIG. 35, FIG. 36, and FIG. 37. Next, IMPRINT-NPs of various compositions were evaluated for their effects on colony forming units (CFUs) after incubation. See FIG. 38, FIG. 39, FIG. 40, FIG. 41.

[0238] PAO1, a laboratory strain of P. aeruginosa, was grown overnight via streak plate in 15 g / L agar at 37 °C. Overnight cultures were prepared by isolating three colonies and inoculating in 1.5 mF EB (lysogeny broth) under shaking conditions at 37°C at 250 rpm for 18 hours. OD600 was measured prior to inoculation. Cultures were then grown in triplicate in EB broth with their respective concentration of metabolite in a shaker set at 37 °C at 250 rpm for 8 hours, so the stationary phase could be met. Cultures were streaked on EB plates, incubated for 24 hours, and CFUs quantified. E. coli (UPEC strain) samples were produced in the same manner. .S'. aureus (N2 strain) was also evaluated. At the end of the study, the plates were imaged and quantified using ImageJ™.

[0239] For soluble metabolites, the concentrations of metabolites were matched based on their composition within nanoparticles based on a 2 mg / mL (FIG. 35) or 1 mg / mE (FIG. 35B through FIG. 35C) concentration of IMPRINT-NPs. For UPEC strain, the most effective metabolites for reducing CFUs were MSA and CTA. ITA reduced the CFU by approximately 40%. CFU rawand normalized data for soluble treatment in S. aureus are shown in FIG. 35 A and FIG. 35B.The results showed that no soluble metabolite treatment reduced N2 CFUs.

[0240] FIG. 36 and FIG. 37 show data for bacterial growth using time-dependent growth curves for treatment with soluble metabolites for UPEC and N2 strains, respectively. Soluble metabolites were not significantly effective in reducing OD600 values for E. coli but had minor decrease post- stationary phase. See FIG. 36. mITA (soluble ITA) was able to slightly reduce N2 bacterial growth while the other metabolites had no impact. See the growth curve in FIG. 37.

[0241] Example 14: Evaluation of IMPRINT-NPs effects on E. coli, P. aeruginosa, and .S'. aureus.

[0242] FIG. 38, FIG. 39, FIG. 40, FIG. 41. show sets of data for IMPRINT-NPs (PLA-, pITA-, pMSA-, pCTA-NPs) and their ability to reduce CFUs when treated at 1 mg / mL on E. coli, P. aeruginosa, and .S'. aureus using methods as described for Example 13.

[0243] FIG. 38 shows results for CFUs for PA01 after incubation of the IMPRINT-NPs. pCTA- NP and pMSA-NP were considerably more effective in reducing each biological replicate, as compared to the control group with no treatment (NT, shown by dotted line).

[0244] FIG. 39 shows the results for total CFU and normalized CFU compared to no treatment for UPEC bacteria incubation with IMPRINT-NPs. A significant reduction in CFUs was observed for PLA-PVA, pITA-NP, and pMSA-NPs. The most effective IMPRINT-NP for reducing UPEC CFUs was pMSA-NP.

[0245] FIG. 40 shows the results for total CFU and normalized CFU compared to no treatment for PAO1 bacteria incubation with IMPRINT-NPs. A significant reduction in CFUs was observed for PLA-PEMA and pCTA-24-NP. The most effective IMPRINT-NP for reducing PAO1 CFUs was pCTA-24-NP, although pMSA-NP also reduced the CFUs but not significantly.

[0246] CFU raw and normalized data for particles in N2 bacteria are shown in FIG. 41 A and FIG. 41B. pITA-NP showed a significant reduction in CFUs along with PLA-PVA, pMSA-NP, pCTA-24-NP, pCTA-48-NP, and pCTA-96-NP. Looking at the normalized CFU data from FIG. 41B, pITA-NP, pCTA-24-NP, and pCTA-48-NP almost completely eliminated N2 CFUs. Other groups were slightly less effective, where pMSA-NP reduced growth by 75%, PLA-PVA NP was capable of almost a 50% reduction, and pCTA-96-NP reduced growth by 80%.

[0247] 4. References.

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Claims

Claims1. A polymer comprising itaconatc (ITA), mcsaconatc (MSA), or citraconatc (CTA).

2. A polymer consisting essentially of ITA, mesaconate MSA, or citraconate CTA.

3. A polymer of claim 2 which is biodegradable.

4. A nanoparticle comprising the polymer of claim 1.

5. A nanoparticle comprising a pharmaceutical composition selected from the group consisting of an ITA polymer, an MSA polymer, and a CTA polymer.

6. A pharmaceutical composition containing the nanoparticle of claim 5.

7. A method of treating an inflammatory condition, traumatic injury, or infectious disease in a subject in need thereof, comprising administering to the subject the polymer of claim 2 or the nanoparticle of claim 5 to the subject.

8. A method of claim 7, wherein the polymer of claim 2 induces CD206 in macrophages or immune cells of the subject.

9. A method of claim 7, wherein the nanoparticles of claim 5 induces CD206 in macrophages or immune cells of the subject.

10. The method of claim 7, wherein the inflammatory condition is selected from the group consisting of cancer, obesity, infectious disease, sepsis, allergic airway disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis, diabetes, lupus, multiple sclerosis, cardiovascular disease, fibrosis and autoimmune diseases.

11. The method of claim 10, wherein the inflammatory condition is obesity.

12. The method of claim 10, wherein the inflammatory condition is allergic airway disease.

13. The method of claim 10, wherein the inflammatory condition is sepsis.

14. The method of claim 10, wherein the infectious disease is selected from the group consisting of a bacterial, viral, or parasitic infection.

15. The method of claim 14 wherein the infectious disease is caused by a Gram-positive bacterium or a Gram-Negative bacterium.

16. The method of claim 14 wherein the infectious disease is caused by COVID- 19.

17. A method of inducing an anti-inflammatory response in a subject suffering from an inflammatory condition comprising administering to the subject the nanoparticle of claim 5.

18. A method of claim 17, wherein the subject suffers from obesity, allergic airway disease, or sepsis.

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