Bioactivities and Anti-Cancer Activities of NKT-Stimulatory Phenyl-Glycolipid Formulated with a PEGylated Lipid Nanocarrier.

Purpose: 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 results, the clinical application of C34 is limited by its poor aqueous solubility. PEGylation enhances the solubility of hydrophobic drugs, and numerous PEGylated drugs have received clinical approval. Consequently, we assessed the biological activity of PEGylated C34 in this study.

Methods: 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.

Results: 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.

Conclusion: 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.