Repurposing an Irritant Botanical: Metabolomic Profiling and Engineered Polymeric Nanoparticles of Euphorbia tirucalli Attenuate Bacterial Virulence and Inflammation in RAW 264.7 Macrophages

Background/Objectives: Targeting bacterial virulence mechanisms and associated inflammation represents a compelling strategy to disarm pathogens without imposing selective survival pressure. While Euphorbia tirucalli (Pencil Cactus) synthesizes potent bioactive secondary metabolites, the extreme irritancy of its raw phytocomplex has historically hindered its biomedical application. This study aimed to overcome these limitations via polymeric nanoencapsulation, leveraging the metabolome of E. tirucalli stem extract to attenuate bacterial virulence and cellular inflammation. Methods: Chemical profiling was conducted via high-resolution LC-Q-Orbitrap HRMS. Polymeric nanoparticles were fabricated using different stabilizers (Tween 80, ethyl cellulose, and PEG 6000) and characterized using TEM, DLS, and XRD. In vitro release kinetics were analyzed in PBS (pH 7.4). Biological evaluations encompassed agar well diffusion, broth microdilution (MIC/MBC), qRT-PCR transcriptional analysis of virulence/biofilm genes in standard bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli), and anti-inflammatory profiling in LPS-stimulated RAW 264.7 murine macrophages via ELISA. Results: HRMS tentatively annotated 46 metabolites, predominantly methoxy flavonoids, flavonoid glycosides, and phenolic acids. The PEG 6000-stabilized nanoformulation achieved optimal colloidal properties (particle size: 65 ± 15 nm, PDI: 0.19 ± 0.03, and zeta potential: −29.6 ± 2.1 mV) alongside sustained Fickian release. Nanoencapsulation markedly suppressed key target genes (toxA, exoS, algD, papC, fimH, and cna;p< 0.05). Broth microdilution confirmed substantial antibacterial enhancement, where the optimized nanoformulation (F3) achieved an 8-fold reduction in MIC/MBC values (15.6 µg/mL vs. 125 µg/mL for crude extract) across S. aureus, E. coli, and P. aeruginosa. In LPS-stimulated RAW 264.7 macrophages, nanoformulations F2 and F3 significantly decreased TLR-2 levels from 5.99 to 5.21 and 5.18, and IL-17 expression from 2.54 to 1.87 and 1.85, respectively. Similarly, NF-κB expression dropped from 3.12 in the LPS group to 2.34 (F2) and 2.27 (F3) (p= 0.0149), confirming pronounced attenuation of acute inflammatory signaling. Conclusions: Polymeric nanoencapsulation of Euphorbia tirucalli extract successfully enhanced antibacterial efficacy and attenuated inflammatory cascades, demonstrating improved in vitro biocompatibility in macrophage cultures. The optimized formulation (F4) achieved an 8-fold reduction in MIC/MBC thresholds (15.6 µg/mL) alongside the downregulation of biofilm and virulence determinants (fimH, papC, and algD). Furthermore, the nanosystems effectively suppressed pro-inflammatory signaling in LPS-stimulated RAW 264.7 macrophages via the TLR-2/NF-κB axis. Overall, this nanoplatform demonstrates improved cellular compatibility and functions as an effective candidate for dual antibacterial and anti-inflammatory interventions.