Melanoma is one of the most immunogenic solid tumors, with strong metastatic characteristics. Although melanoma is curable when detected in a localized form, the long-term survival rates of patients are difficult to improve by any single therapy. The combination of photodynamic therapy (PDT) and immunotherapy has shown encouraging therapeutic efficacy against various cancers due to PDT-induced immunogenic cell death. Natural killer (NK) cell-based immunotherapy has the ability to enhance the anticancer effects of PDT. The NK cell family 2 member D/ligand (NKG2D/NKG2DL) pathway acts as a master switch in the activation of NK cells and mediates the recognition of tumor cells by NK cells. However, tumor cells can produce soluble NKG2DLs via shedding, which mediates the immune escape of tumors. Regulating NKG2D-related molecular pathways can effectively improve the anti-tumor efficacy of NK cells. Further, combined with PDT, NK-related anti-tumor immunity and therapeutic effects could be significantly promoted. In this study, a tumor microenvironment- and light-responsive nanoparticle—MMP-2-responsive polypeptide mixed liposome (MRPL-SC)—was designed to realize synergistic anti-tumor effects of Ce6-mediated PDT and SB-3CT enhanced NKG2D-based immunotherapy, and spatial–temporally controlled release of loaded drugs.

To synthesize MRPL-SC, DSPE-PEG(3400)-MRP-β-CD with an MMP-2 cleavable sequence was formed via multistep reactions. Then, MRPL-SC, combined with β-cyclodextrin (β-CD) containing SB-3CT to suppress the overexpressed MMP-2 that is tightly associated with the tumor NK-related escape mechanism in the tumor microenvironment and photosensitizer Ce6-loaded liposomes, was prepared using the film dispersion and extrusion method. The multi-responsive release ability of SB-3CT triggered by the MMP-2 abundant tumor microenvironment and chlorin e6 (Ce6) by 660 nm laser irradiation was studied in vitro. Cellular uptake, ROS generation, and the synergistic anti-tumor effects of MRPL-SC-mediated PDT and enhanced NK immunotherapy were studied in A375 cells, followed by the analysis of NKG2DLs expression. In addition, the anti-tumor efficiency and safety of MRPL-SCs were studied in tumor-bearing nude BALB/c mouse models.

The prepared MRPL-SC exhibited a typical liposome structure with a phospholipid bilayer and formed single/double-layer vesicles for drug encapsulating and intracellular delivering with a particle size about 160-170 nm (Fig. 2). Then, the MMP-2-responsive polypeptide linker was hydrolyzed by MMP-2, and SB-3CT encapsulated in β-CD was rapidly released, with accumulation reaching 72.26% after 240 min, indicating the responsiveness of MRPL-SC to the MMP-2 abundant tumor extracellular microenvironment. The released SB-3CT effectively regulated the expression of NKG2DLs and generation of soluble NKG2DLs in tumor tissues by directly inhibiting MMP-2 in situ. Ce6-encapsulated liposomes entered the tumor cells through endocytosis and induced severe cytotoxicity by PDT under 660 nm laser irradiation (Fig.5). In addition, Ce6-PDT also induced NKG2DLs expression, thereby realizing spatiotemporal synergistic enhancement of NK immunity in tumor tissues (Fig. 6). By employing the A375 tumor-bearing nude mice xenograft model, MRPL-SCs showed higher amounts of aggregation in tumor tissue and efficiently suppressed melanoma growth after laser irradiation (Fig. 8) without significant cytotoxic effects in the major organs.

In this study, a novel multi-responsive nanoliposome composed of photosensitizer Ce6-liposomes and MMP-2 antagonist SB-3CT linked by MMP-2 responsive peptide was successfully fabricated via film dispersion and extrusion. The synergistic anti-tumor effect of MRPL-SC-mediated PDT and NK cell immune enhancement was observed in both melanoma cells and nude mouse xenograft models. This study not only developed a new multi-responsive nanocomplex for better integration of PDT and immunotherapy but also elucidated a novel paradigm for NK cell-mediated photodynamic immunotherapy for melanoma.