DOI: 10.25413/sun.25226348.v1
title: The influence of biomimetic nanoparticles on macrophage phenotype and their anti-mycobacterial efficacy on macrophages.

description: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) remains a major public health concern. Despite the availability of various treatment regimens, treatment failure is common, partly attributed to the phenomenon of bacterial persisters. In addition, repeated or lengthy treatments can potentially induce drugresistant strains of M. tuberculosis which further compound TB control. New approaches to TB therapy are therefore needed. To promote the intracellular killing of M. tuberculosis through pro-inflammatory responses (M1), a promising approach is the application of nanoparticles (NPs), which include lipid-polymer hybrid (LPN), to deliver drugs or immunomodulatory compounds such as Curdlan (CN) to macrophages. This represents a novel, host-based therapeutic approach for TB. While M1 polarized macrophages are thought to be the most effective in controlling M. tuberculosis infection, it has been shown that intracellular bacilli induce a shift to the M2 phenotype. There is no evidence that the introduction of NPs such as LPN conjugated with CN can induce a shift in macrophage phenotype or that the macrophage polarization state affects the antibacterial activity of NPs. In this study, we first set out to evaluate the anti-mycobacterial efficiency of CN-functionalized NPs in vitro, which had previously shown to be promising in the inhibition of mycobacterial growth intracellularly and in vivo in a parent study. Primary cells derived from the C57BL/6 mice bone marrow called bone marrow derived macrophages (BMDMs) were used for this study. We used an attenuated M. tuberculosis H37Rv laboratory strain with a reporter plasmid (pMV306hsp+Lux) to allow real-time monitoring of luminescence as a proxy for the bacterial load. Results from the real-time luminescence monitoring confirmed that the CNfunctionalized NPs is a good anti-mycobacteria agent as previously demonstrated in the parent study. We further explored if macrophage phenotypic state and function might alter upon treatment with selected NPs, and in turn, if macrophage phenotype alters the immunological response induced by selected NPs. In our study, we examined macrophage phenotype through multiplex cytokine analysis, focusing on 11 analytes indicative of macrophage polarization status. We utilized a specific mathematical formula, termed the macrophage polarization ratio (MPR), designed for this investigation. Results demonstrated that macrophages have an attraction for CN-functionalized NPs. In addition, the NPs have an impact on macrophage immunologic and phenotypic activity. The NPs induced high production of pro-inflammatory cytokines such IFN-ɣ, IL-1β, IL-12p70, IL-18, IL-2, IL-5, TNF-α, and IL-6. In summary, our study demonstrated the bacterial strain's is consistent in terms of replication homogeneity for both liquid culture and intracellularly, confirming M. tuberculosis's suitability for TB research with a reporter plasmid (pMV306hsp+Lux). Significantly, the CN-functionalized nanoparticles demonstrated noteworthy anti-mycobacterial efficacy. Data on macrophage viability highlighted the possible importance of a lower multiplicity of infection (MOI) for BMDMs. Recognizing the need for additional research, our findings indicated that CN-functionalized nanoparticles have a discernible influence on macrophage phenotype, specifically by increasing the expression of M1 markers. This comprehensive understanding highlights the promising role of CN-functionalized nanoparticles in shaping macrophage responses, implying avenues for targeted tuberculosis therapeutic interventions. By investigating the complex area of nanoparticles' impact on macrophage phenotype during TB treatment, this study filled a knowledge gap. This study expands our understanding of the dynamics of TB treatment and emphasizes the need for additional research to fully realize the promise of nanoparticle-mediated interventions in the treatment of TB. However, further research into the concentration-dependent anti-mycobacterial activity and transcriptomic response is required.