Abstract Body: Erectile dysfunction (ED) in obesity is closely linked to chronic inflammation, immune activation, and endothelial dysfunction. Macrophages play a key role in this process, with Toll-like receptor 9 (TLR9) acting as a critical mediator. In obesity, elevated levels of circulating cell-free DNA function as damage-associated molecular patterns, activating TLR9 in macrophages and promoting the release of pro-inflammatory cytokines such as TNF-α and IL-6, which may impair erectile function. We hypothesized that TLR9 activation in macrophages exacerbates the inflammatory profile and contributes to vascular dysfunction involved in ED, and that the targeted inhibition of this pathway could be therapeutically beneficial. To investigate this, we developed poly(lactide-co-glycolide) (PLG) microparticles designed to be engulfed by macrophages via surface presentation of phosphatidylserine (PS:PLG), acting as carriers for TLR9-targeting agents. Fluorescence microscopy and flow cytometry revealed that PLG microparticles alone were poorly internalized by macrophages, with uptake observed in only 18.1% of cells, whereas PS-coated PLG (PS:PLG) particles significantly enhanced internalization, reaching 70.1% of cells. In LPS-stimulated macrophages, PS:PLG reduced in ~50% the secretion of TNF-α and IL-6 and increased in ~50% IL-10 levels, indicating an anti-inflammatory shift. When loaded with the TLR9 agonist, ODN2395, PS:PLG microparticles enhanced the pro-inflammatory cytokine release, confirming its selective targeting and delivery efficiency. To assess functional effects, isolated corpus cavernosum, aorta, and pudendal artery from C57BL/6J mice were co-incubated with macrophages and ODN2395. After 24 hours, tissues were mounted in myographs to assess vascular reactivity. ODN2395 increased phenylephrine-induced maximal contraction in the pudendal artery (Emax control: 8.11 ± 0.77 mN vs ODN2395: 12.51 ± 1.22 mN) and corpus cavernosum (Emax control: 0.8 ± 0.11 mN vs ODN2395: 1.5 ± 0.06 mN) independent of macrophage presence. No significant changes were observed in aorta. These findings suggest that TLR9 activation contributes to a pro-inflammatory phenotype and vascular dysfunction relevant to ED. Therefore, in vivo experiments are needed to support the potential of PS:PLG microparticles to selectively target macrophages and modulate TLR9-driven inflammation as a promising approach for the treatment of immune-mediated erectile dysfunction in obesity.