Itaconate microparticles for intracellular targeting of macrophage inflammation

Introduction: Chronic inflammation is a key driving factor in nearly all non-communicable diseases (NCDs). Macrophages play a critical role in the initiation, maintenance, and resolution of inflammation, making them a pivotal target for chronic NCDs. Recent advances have highlighted that macrophage metabolic intermediates (metabolites) play a role in keeping inflammation in check; itaconate (IA) is a macrophage metabolite that regulates inflammation intracellularly through multiple mechanisms. IA is an attractive biomimetic therapy, but delivering effective concentrations to macrophages remains a major barrier due to its rapid rate of circulatory removal and limited IA cell permeability.

IA-based polyester polymer materials previously developed by our lab (Davenport Huyer et al, Adv. Func. Mat 2021) achieve local concentrated delivery by incorporating IA directly into their molecular structure, and relying on the controlled polymer degradation to regulate macrophage inflammation. A unique macrophage immune function is internalizing micron sized foreign material through phagocytosis; these particles could harness this property to deliver IA polymers inside the cell and release IA, overcoming cell permeability limitations while specifically targeting macrophages. Polymer microparticles (MPs) are thus a promising vehicle for intracellular delivery of metabolites through mechanisms of controllable polymer degradation. In this work, we describe the fabrication of itaconate-based polymer microparticles (IA-MP) and their ability to target macrophage inflammation through intracellular phagocytotic delivery mechanisms (Fig. 1A).

Materials and

Methods: IA polymers were synthesized through polycondensation reactions of dimethyl itaconate with 1,12-dodecandiol (1:1.1 molar ratio) to produce polymers of varied material properties. Polymer molecular weight (MW) and co-monomer structure were characterized using gel permeation chromatography (GPC) and proton nuclear magnetic resonance (H-NMR). IA-MPs were fabricated using a water in oil in water emulsion method, with variation in homogenization speed to control particle size, and assessed through scanning electron microscopy (SEM). Correlation of IA-MP material formulation to degradation was determined under hydrolytic (biologically relevant) and enzymatic conditions that mimic macrophage phagocytosis to assess short term (1-48 hr) IA release relevant to intracellular delivery timelines.

Murine bone marrow derived macrophages (BMDM) isolated from C57BL6 bone marrow monocytes were cultured for 6 days with macrophage colony stimulating factor (M-CSF, 20ng/mL). IA-MP particle uptake kinetics (rhodamine loaded) was quantified through sequential live cell imaging over 24h at varying particle concentrations (0.5-0.005 mg/million cells). Toxicity was assessed at endpoint (24hr) using SYTOX staining, against a Hoechst nuclear counterstain, and analyzed with Gen5 software. Macrophages were stimulated with LPS and treated with IA-MPs to understand inflammatory behaviour over 24h of particle treatment, in comparison to stimulated and unstimulated particle free controls, and non degradable polystyrene control particles. Reverse transcriptase-polymerase chain reaction (RT-qPCR) and ELISA was used to correlate inflammatory gene (e.g. Il1b, Hmox1, Nos2) and protein (Il1b, IL6, TNFα) expression to polymer material properties and degradation rate. Flow cytometry was applied to correlate individual macrophage particle uptake (rhodamine) with changes in macrophage activation (CD86, MHCII, NOS2).

Results, Conclusions, and Discussions: Dimethyl itaconate was reacted with 1,12-dodecanediol with varied reaction duration and pressure to produce IA polymers with number-average molecular weights (Mn) of 13 kDa (high MW) and 2 kDa (low MW) that were formulated into IA-MP (Fig. 1B). Degradation of IA polymers released IA and associated oligomers under physiologically relevant (37°C, pH7.4) degradation conditions. Particles were successfully internalized by BMDM (Fig. 1C), exhibiting increased uptake efficiency with increasing particle concentration. IA-MPs with a concentration of 0.1 mg/million cells in LPS stimulated BMDM exhibited 72% uptake of particles (24 hr treatment) with corresponding 13% cell death.

LPS stimulated macrophages treated (24hr) with high and low MW IA-MPs showed significant downregulation of Il6 and Il12, significant upregulation of Hmox1, and no significance in TNFα with respect to LPS stimulated cells without particle treatment. This gene expression regulation of Il6 (Fig. 1D), Hmox1, TNFα, and Il12 is consistent with regulatory pathways associated with exogenous IA, highlighted by similar regulation to soluble IA (5mM) and membrane permeable 4-octyl itaconate (125uM) in our studies. IA particles with low MW provided stronger regulation of inflammatory pathways with respect to all genes of interest, which is consistent with increased intracellular polymer degradation related to variations in material properties (molecular weight). Preliminary flow cytometry analysis illustrates that IA-MP containing BMDM reduce MHCII expression in comparison to non-particle containing stimulated macrophages, suggesting downregulated inflammatory behaviour is specific to particle internalization.

Through this proof-of-concept work, we have demonstrated that the intracellular IA-MP based delivery of itaconate has potential as an effective targeted therapeutic strategy against chronic macrophage inflammation, eliminating current exogenous dosage limitations of this exciting biomimetic therapy. The application of IA-MPs could pave the way for innovative, metabolite-based microparticle drug delivery systems for the treatment of inflammatory disease.

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