Mannose-functionalized liposomal delivery of levamisole and lipopolysaccharide enhances therapeutic responses through tumor-associated macrophage modulation in colon cancer

Immunotherapeutics offer promise for colon cancer treatment but are often limited by tumor heterogeneity. This study reports the development of mannose-functionalized (Mannosylated) liposomes co-encapsulating Levamisole (LEV) and Lipopolysaccharide (LPS) that targets M2-Tumor associated macrophages (TAMs) and enhance therapeutic efficacy. Liposomes were prepared by thin-film hydration method and optimized using Box-Behnken design. FT-IR analysis confirmed mannose conjugation at 1644 cm−1, indicating amide bond formation. The optimized formulation exhibited a particle size of 169.5 ± 0.71 nm, encapsulation efficiencies of 50.28 ± 2.64% (LEV) and 95.76 ± 0.10% (LPS) and demonstrated controlled drug release of LEV in vitro at pH 1.2, 6.8, and 7.4 conditions. In vivo evaluation in CT26 orthotopic colon tumor model showed enhanced tumor localization, significant tumor regression and improved survival outcomes in formulation treated group when combined with 5-fluorouracil (5-FU). qRT-PCR analysis revealed downregulation of M2 macrophage markers (CD206, Arg1). Phagocytosis assay demonstrated significantly higher phagocytic clearance (p < 0.05). Although the delayed-type hypersensitivity (DTH) assay showed a decline in % DTH response, histopathology of spleen and thymus implied enhanced lymphocyte cellularity with acute inflammatory infiltrations confirming elevated immune activation. The Mannosylated liposomal system effectively delivers immunomodulators to modulate the tumor microenvironment and enhance 5-FU based therapy in colon cancer.