Fig. 11

(a) Cytokine Excretion Group (ELISA): Cytokine release levels (TNF-α, IL-6, and IL-1β) assessed in various treatment groups using ELISA. Cells treated with LPS (positive control) exhibited markedly increased cytokine levels (LPS induced high TNF-α (194.3 ± 8.5 pg/mL), IL-6 (242.2 ± 10.4 pg/mL), and IL-1β (182.1 ± 7.2 pg/mL) (P < 0.0001)), suggesting robust immune activation. Unbound MTX caused a moderate release of cytokines due to oxidative stress and cell death, whereas MTX-SLNs and MTX-LF-SLNs significantly lowered cytokine production((P < 0.0001), indicating enhanced biocompatibility and a diminished inflammatory reaction. (b) Images of Cell Morphology: Images of representative cell morphology for each treatment group. Black arrows show alterations in cellular structure. Control (untreated) cells exhibit typical morphology, whereas LPS-treated cells show indications of inflammation. Free MTX therapy causes significant cellular stress, while MTX-SLNs and MTX-LF-SLNs demonstrate better cell integrity, suggesting diminished cytotoxic impacts. (c) Flow Cytometry Analysis of ICAM-1 & HLA-DR Expression. Flow cytometry histograms show that LPS treatment strongly upregulated ICAM-1 (82.3 ± 6.5%) and HLA-DR (90.7 ± 7.2%), indicating immune activation. Free MTX moderately increased these markers, while MTX-SLNs (ICAM-1: 13.2 ± 4.2%, HLA-DR: 15.5 ± 3.2%) and MTX-LF-SLNs (ICAM-1: 14.2 ± 4.2%, HLA-DR: 12.3 ± 3.4%) significantly suppressed their expression (P < 0.0001), demonstrating reduced immunogenicity. (d). Activation of the Immune System (Analysis via Flow Cytometry). LPS treatment triggered robust immune activation, notably increasing ICAM-1 (82.3 ± 6.5%) and HLA-DR (90.7 ± 7.2%) (P < 0.0001). Free MTX moderately elevated these markers, whereas MTX-SLNs and MTX-LF-SLNs significantly downregulated ICAM-1 and HLA-DR (P < 0.0001), suggesting improved pharmacological safety and diminished immunogenic response.