Non-Genetic Reprogramming of Monocytes via Microparticle Phagocytosis for Sustained Modulation of Macrophage Phenotype

Abstract

Monocyte-derived macrophages orchestrate tissue regeneration by homing to sites of injury, phagocytosing pathological debris, and stimulating other cell types to repair the tissue. Accordingly, monocytes have been investigated as a translational and potent source for cell therapy, but their utility has been hampered by their rapid acquisition of a pro-inflammatory phenotype in response to the inflammatory injury microenvironment. To overcome this problem, we designed a cell therapy strategy where we collect and exogenously reprogram monocytes by intracellularly loading the cells with biodegradable microparticles containing an anti-inflammatory drug in order to modulate and maintain an anti-inflammatory phenotype over time. To test this concept, poly(lactic-co-glycolic) acid microparticles were loaded with the anti-inflammatory drug dexamethasone (Dex) and administered to primary human monocytes for four hours to facilitate phagocytic uptake. After removal of non-phagocytosed microparticles, microparticle-loaded monocytes differentiated into macrophages and stored the microparticles intracellularly for several weeks in vitro, releasing drug into the extracellular environment over time. Cells loaded with intracellular Dex microparticles showed decreased expression and secretion of inflammatory factors even in the presence of pro-inflammatory stimuli up to 7 days after microparticle uptake compared to untreated cells or cells loaded with blank microparticles. This study represents a new strategy for long-term maintenance of anti-inflammatory macrophage phenotype using a translational monocyte-based cell therapy strategy without the use of genetic modification. Because of the ubiquitous nature of monocytederived macrophage involvement in pathology and regeneration, this strategy holds potential as a treatment for a vast number of diseases and disorders. RESULTS Microparticle Characteristics and Intracellular StabilityPoly(lactic-co-glycolic) acid (PLGA) microparticles were fabricated with dexamethasone (Dex) or with tetramethylrhodamine (TRITC) as a fluorescent model drug. Microparticles ranged in diameter from 0.98 to 2.05 µm with polydispersity indices between 0.08 and 0.28 ( Fig. 2A).Microparticles were administered to primary human monocytes for 4 hours followed by removal of non-phagocytosed microparticles. Thereafter, monocytes were cultured in macrophage colony stimulating factor (MCSF)-containing media to induce differentiation into macrophages and were imaged at regular intervals (Fig. 2B). Cell area increased over time for both untreated and microparticle-loaded cells, indicating that that monocyte-to-macrophage differentiation was not hindered by intracellular microparticle loading (Fig. 2C). Intracellular fluorescent microparticles were detected for up to 16 days in vitro (detection and quantification methods outlined in Sup. Fig. 1). Interestingly, the number of microparticles ( Fig. 2D) and their intensity per cell ( Fig. 2E) increased in the first three days, which may be due to cell death, phagocytosi...