TNFR2-Specific P20 Peptide-Conjugated Lipid Nanoprobe Enables Dual-Modal PET/CT Molecular Imaging for Precise Detection of Colon Cancer

Background: Tumor necrosis factor receptor 2 (TNFR2) is a pivotal immune checkpoint that is highly expressed within the immunosuppressive tumor microenvironment (TME), particularly on regulatory T cells (Tregs). The P20 peptide specifically binds TNFR2, but its rapid enzymatic degradation in vivo severely limits its clinical translation as an imaging tracer. Methods: Here, we developed a P20-conjugated lipid nanoprobe (DPP) via thin-film hydration and systematically evaluated its physicochemical properties, receptor-binding specificity, biosafety, and in vivo positron emission tomography (PET)/computed tomography (CT) imaging performance in male C57BL/6J mice bearing subcutaneous MC38 wild-type (WT) or TNFR2-knockout (KO) colon tumors. Results: DPP exhibited a near-spherical morphology, a predominant particle-size distribution peak at approximately 125.6 nm, a negative surface charge, and relatively stable hydrodynamic diameters over 7 days. In vitro assays supported TNFR2-associated cellular recognition and showed negligible cytotoxicity under the tested conditions. Micro-PET/CT imaging revealed that DPP markedly improved tumor retention (tumor SUVmean at 4 h: 0.577 ± 0.314) compared with free P20 (SUVmean at 4 h: 0.065 ± 0.011; n = 6 per group; two-sided Welch’s unpaired t-test with Benjamini–Hochberg correction, adjusted q = 0.010298) in MC38-WT tumors. Tumor SUVmean was higher in WT than in KO tumors at 7 h (0.677 ± 0.181 vs. 0.381 ± 0.191; p = 0.02942) and 12 h (0.428 ± 0.121 vs. 0.224 ± 0.108; p = 0.01588), with both adjusted q values equal to 0.05885 (n = 6 and n = 5, respectively). Notably, tumor-to-muscle uptake ratios were also higher in WT tumors at 7 h (13.11 ± 5.10 vs. 6.86 ± 2.97; p = 0.03459; adjusted q = 0.13836). Conclusions: These results demonstrate that the liposomal formulation enhances tumor-associated uptake of the P20-based tracer, while TNFR2-dependent uptake and the non-invasive monitoring of Treg compartments remain to be validated.