Tumor-associated macrophages (TAMs) are a promising therapeutic target for cancer immunotherapy. Targeted delivery of therapeutic drugs to the tumor-promoting M2-like TAMs is challenging. Here, we developed M2-like TAM dual-targeting nanoparticles (M2NPs), whose structure and function were controlled by α-peptide (a scavenger receptor B type 1 (SR-B1) targeting peptide) linked with M2pep (an M2 macrophage binding peptide). By loading anti-colony stimulating factor-1 receptor (anti-CSF-1R) small interfering RNA (siRNA) on the M2NPs, we developed a molecular-targeted immunotherapeutic approach to specifically block the survival signal of M2-like TAMs and deplete them from melanoma tumors. We confirmed the validity of SR-B1 for M2-like TAM targeting and demonstrated the synergistic effect of the two targeting units (α-peptide and M2pep) in the fusion peptide (α-M2pep). After being administered to tumor-bearing mice, M2NPs had higher affinity to M2-like TAMs than to tissue-resident macrophages in liver, spleen, and lung. Compared with control treatment groups, M2NP-based siRNA delivery resulted in a dramatic elimination of M2-like TAMs (52%), decreased tumor size (87%), and prolonged survival. Additionally, this molecular-targeted strategy inhibited immunosuppressive IL-10 and TGF-β production and increased immunostimulatory cytokines (IL-12 and IFN-γ) expression and CD8 T cell infiltration (2.9-fold) in the tumor microenvironment. Moreover, the siRNA-carrying M2NPs down-regulated expression of the exhaustion markers (PD-1 and Tim-3) on the infiltrating CD8 T cells and stimulated their IFN-γ secretion (6.2-fold), indicating the restoration of T cell immune function. Thus, the dual-targeting property of M2NPs combined with RNA interference provides a potential strategy of molecular-targeted cancer immunotherapy for clinical application.

Liver sinusoidal endothelial cells (LSECs) are responsible for the immunologic tolerance of liver which is a common site for visceral metastases, suggesting its potential role as an target for cancer immunotherapy. However, targeted modulation of LSECs is still not achieved thus far. Here, we report LSECs are specifically targeted and modulated by melittin nanoparticles (α-melittin-NPs). Intravital imaging shows that LSECs fluoresce within 20 s after intravenous injection of α-melittin-NPs. α-melittin-NPs trigger the activation of LSECs and lead to dramatic changes of cytokine/chemokine milieu in the liver, which switches the hepatic immunologic environment to the activated state. As a result, α-melittin-NPs resist the formation of metastatic lesions with high efficiency. More strikingly, the survival rate reaches 80% in the spontaneous liver metastatic tumor model. Our research provides support for the use of α-melittin-NPs to break LSEC-mediated immunologic tolerance, which opens an avenue to control liver metastasis through the immunomodulation of LSECs.

Kupffer cells (KCs), potent scavenger cells located in hepatic sinusoids, constantly phagocytize and degrade foreign materials to maintain metabolism and clearance. Understanding the strategic KC arrangement which links to their spatial location and function in hepatic lobules, the basic functional unit in the liver, is highly valuable for characterizing liver function. However, selectively labeling KCs and characterizing their function in vivo remains challenging. Herein, a fast self-assembled pomegranate structure-like nanoparticle with "nanopomegranate seeds" of dye aggregates has been developed, which has dual-modality "off/on" capability. This nanopomegranate shows good photostability, a high extinction coefficient, a high KC labeling efficiency (98.8%), and better visualization of KC morphology than commercial FluoSpheres. In vivo photoacoustic (PA) and fluorescence imaging consistently visualize that KCs are strategically distributed along the central vein (CV)−portal triad (PT) axis in each liver lobule: more and larger KCs exist in areas closer to the PTs. The high-resolution PA quantitative data further revealed that the density of KCs was linearly dependent on the r n /r max ratio (their relative location along the CV−PT axis) (R 2 = 0.7513), and the KC density at the outermost layer is almost 246-fold that at the innermost layer (each layer is 8 μm). Notably, the phagocytic ability of KCs located in layers with r n /r max ratios of 0.167−0.3 varies in a zigzag pattern, as evidenced by their different PA intensities. Additionally, the fluorescence imaging quantitation suggests similar fluorescence activation of nanopomegranate in KCs. Nanopomegranates combined with dual-modality imaging reveal the strategic arrangement of KCs in vivo, greatly extending our understanding of liver physiology.