Bioengineered extracellular vesicles escape lysosomal degradation and deliver Tet-PKM2 for macrophage immunometabolic reprogramming and periodontitis treatment

Modulating macrophage phenotype and function via immunometabolic reprogramming represents a new therapeutic paradigm to combat chronic inflammatory diseases such as periodontitis. Tetrameric pyruvate kinase M2 (Tet-PKM2), a highly active metabolic enzyme involved in the flux of glucose-derived carbons into the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), was found to be dramatically decreased in response to inflammation, rendering a potential immunometabolic target for developping new therapeutics. Hence, we report a large extracellular vesicle (LEV) that is bioengineered to intracellularly deliver Tet-PKM2 for the reprogramming of proinflammatory macrophages and the restoration of their aberrant immunometabolism. We engineered Tet-PKM2-enriched LEVs modified by tannic acid (LEVs Tet−PKM2 @TA) that can intracellularly deliver Tet-PKM2 and increase their ability to escape lysosomal degradation for the intracellular delivery of Tet-PKM2. In vitro , LEVs Tet−PKM2 @TA were able to rescue aberrant pyruvate metabolism in lipopolysaccharide (LPS)-activated macrophages by increasing TCA cycle activity and enhancing mitochondrial OXPHOS metabolism. In vivo , LEVs Tet−PKM2 @TA exerted robust immunomodulatory effects by increasing pyruvate kinase (PK) activity and coaxing macrophages toward the M2 phenotype, ultimately resulting in robust periodontal tissue regeneration in a mouse ligature-induced periodontitis model. This study provides a versatile and safe method for the targeted delivery of Tet-PKM2 via EVs to modulate macrophage phenotype and function. Our work demonstrates a new concept for immunometabolic reprogramming to treat chronic inflammatory diseases. Schematic illustration of (A) the yield of bioengineered extracellular vesicles (LEVs Tet−PKM2 @TA) and (B) following LEVs Tet−PKM2 @TA uptake, cellular events (lysosomal degradation escape and cell immunometabolic reprogramming) occurred within macrophages. The up (down) arrows indicate increases (decreases) in the corresponding molecules. Abbreviations: Tet-PKM2, tetrameric pyruvate kinase M2; LEV, large extracellular vesicle; TA, tannic acid; PEP, phosphoenolpyruvate; LA, lactate; MMP, mitochondrial membrane potential; ATP, adenosine triphosphate; TCA, tricarboxylic acid; OXPHOS, oxidative phosphorylation. • Tet-PKM2 downregulation causes aberrant immunometabolism and hyperinflammation in gingival macrophages during periodontitis. • TEPP-46 treatment of PKM2-overexpressing cells enables enrichment of Tet-PKM2 in their large extracellular vesicles (LEVs). • Tannic acid (TA) modification of Tet-PKM2-enriched LEVs (LEVs Tet−PKM2 @TA) can achieve lysosomal escape within macrophages. • LEVs Tet−PKM2 @TA coax LPS-activated M1 macrophages toward anti-inflammatory phenotypes through metabolic reprogramming. • LEVs Tet−PKM2 @TA combat periodontitis via immunometabolic reprogramming and promotion of M2 polarization in vivo.