In vitro evaluation of vismodegib-loaded anti-PD-L1 stealth immunoliposomes for melanoma treatment

Skin cancers are among the most prevalent malignancies worldwide and arise from a combination of genetic predisposition and environmental factors, including aging and ultraviolet radiation exposure. They are broadly classified into non-melanoma skin cancers, including basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma, and cutaneous melanoma, the most aggressive form due to its high metastatic potential. Several skin tumors show increased expression of programmed death-ligand 1 (PD-L1), an immune checkpoint protein involved in tumor immune evasion. In addition, aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in skin cancer biology, particularly in BCC and, to a lesser extent, in melanoma progression. Therefore, simultaneous targeting of PD-L1 and the Hh pathway may represent a promising therapeutic strategy. Although anti-PD-L1 immunotherapies and Hh pathway inhibitors are available, they are usually administered as separate treatments. In this context, our group developed vismodegib-loaded immunoliposomes functionalized with the Fab’ fragment of atezolizumab, an anti-PD-L1 monoclonal antibody. Vismodegib is a selective inhibitor of Smoothened (SMO), a key component of the Hh pathway. This dual-targeted nanosystem was designed to enhance drug delivery to PD-L1-expressing tumor cells, reduce the systemic toxicity associated with free vismodegib, and potentially contribute to PD-L1 blockade at the tumor cell surface. The present project focused on the in vitro evaluation of this nanoformulation. Vismodegib was incorporated into the liposomal bilayer, while the Fab’ fragment was conjugated to the liposome surface. The formulation was characterized by dynamic light scattering, zeta potential measurements, RP-HPLC, SDS-PAGE, and micro-BCA assay. Biological performance was evaluated using HaCaT human keratinocytes as a non-tumorigenic control model and SK-Mel-28 human melanoma cells as the tumor model. PD-L1 expression was assessed by flow cytometry, cytotoxicity by ATPlite assay, and cellular uptake by confocal microscopy in both 2D cultures and spheroids. Spheroid growth and morphology were also monitored, while GLI-1 expression, a downstream marker of Hh pathway activation, was analyzed by Western blot. The results showed that PD-L1-targeted immunoliposomes exerted higher antitumor activity in melanoma cells than free vismodegib and non-targeted liposomes. Confocal microscopy suggested enhanced uptake in SK-Mel-28 cells upon active targeting, while 3D spheroid studies showed that the formulations could interact with and partially penetrate the tumor-like structure. Moreover, Western blot analysis revealed a reduction in GLI-1 expression after treatment with the immunoliposomes, suggesting modulation of the Hh pathway. Overall, these findings support the potential of PD-L1-targeted vismodegib-loaded immunoliposomes as a promising nanomedicine-based strategy for melanoma treatment.