These findings demonstrate that the newly formulated PLN-C34 retains NKT-stimulatory activity and anti-cancer efficacy of C34, supporting the potential of PLN as a solvent for C34 for further development in cancer therapy.

Murine NK1.2 cells were cultured with A20-CD1d cells in the presence of either PEGylated lipid nanocarriers encapsulating C34 (PLN-C34) or C34 dissolved in DMSO to determine IL-2 production via ELISA. C57BL/6 mice were i.v. injected with C34 or PLN-C34 to examine cytokine profiles and immune cell populations using luminex and flow cytometry, respectively. The anticancer effects of C34 and PLN-C34 were evaluated in mice bearing TC-1 lung cancer and B16 melanoma tumors. Additionally, human PBMCs were cultured with C34 or PLN-C34 to measure cytokine production through luminex.

The glycolipid α-galactosylceramide (α-GalCer), when presented by CD1d, can modulate the immune system through the activation of natural killer T (NKT) cells. Previously, we synthesized over 30 analogs of α-GalCer and identified a compound, C34, which features two phenyl rings on the acyl chain. C34 exhibited the most potent NKT-stimulating activities, characterized by strong Th1-biased cytokines and potent anti-tumor effects in several murine tumor models. Importantly, unlike α-GalCer, C34 did not induce NKT cell anergy. Despite these promising

PLN-C34 demonstrated a comparable capacity to C34 in activating the NKT cell line in vitro and inducing various cytokines in vivo. Furthermore, treatment with either PLN-C34 or C34 significantly prolonged the survival of TC-1- and B16F10-bearing mice to a similar extent. Additionally, PLN-C34 effectively stimulated cytokine responses in human NKT cells, comparable to those induced by C34.