Targeted Fusogenic Liposomes for Effective Tumor Delivery and Penetration of Lipophilic Cargoes.

Nanoparticle-based drug delivery has been widely used for effective anticancer treatment. However, a key challenge restricting the efficacy of nanotherapeutics is limited tissue penetration within solid tumors. Here, we report a targeted fusogenic liposome (TFL) that can selectively deliver lipophilic cargo to the plasma membranes of tumor cells. TFL is prepared by directly attaching tumor-targeting peptides to the surface of FL instead of the cationic moieties. The lipophilic cargo loaded in the membrane of TFL is transferred to the plasma membranes of tumor cells and subsequently packaged in the extracellular vesicles (EVs) released by the cells. Systemically administered TFL accumulates in the perivascular region of tumors, where the lipophilic cargo is unloaded to the tumor cell membranes and distributed autonomously throughout the tumor tissue via extracellular vesicle-mediated intercellular transfer. When loaded with a lipophilic pro-apoptotic drug, thapsigargin (Tg), TFL significantly inhibits tumor growth in a mouse colorectal cancer model. Furthermore, the combination treatment with TFL (Tg) potentiates the antitumor efficacy of FDA-approved liposomal doxorubicin, whose therapeutic effect is limited to perivascular regions without significant toxicity.

Management of lymph node metastasis via local chemotherapy can prevent distant metastasis and improve survival in mice.

Management of lymph node metastasis (LNM) by conventional modalities such as radiotherapy and systemic chemotherapy exhibit limited LNM selectivity and therefore can cause off-target adverse events. While development of LNM-specific drug delivery systems has tremendous potential to provide a safer treatment modality and improve cancer treatment, precise assessment of therapeutic efficacy and implications has been challenging due to lack of a suitable preclinical model. Here, we established an experimental LNM model in mice by directly seeding cancer cells into a lymph node (LN), which developed spontaneous LNM-borne distant metastasis (DM) in the absence of a primary tumor. In the model, early, but not late, management of LNM before thereof tumor cells systemically disseminated could confer significant survival benefit, which suggests that time to LNM management is critical. Systematic comparative assessment of various local drug delivery systems revealed that a micellar formulation could achieve highly LNM-specific delivery of a chemotherapeutic agent, which was superior to systemic chemotherapy, effective at a very low dose, and safe. This study suggests not only that the experimental LNM model provides a useful preclinical model to study LNM management and its therapeutic implications but also that micelles are a promising drug delivery system for LNM management via local administration.

Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma.

Isolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

Phage display-identified PD-L1-binding peptides reinvigorate T-cell activity and inhibit tumor progression.

Blockade of programmed cell death ligand-1 (PD-L1) restores T-cell activity and enhances anti-tumor immunity. Screening a phage-displayed peptide library for peptides that selectively bind to PD-L1-overexpressing cells identified two peptides, CLQKTPKQC and CVRARTR (PD-L1Pep-1 and PD-L1Pep-2, respectively) that appeared to block PD-L1. PD-L1Pep-1 and PD-L1Pep-2 preferentially bound to high PD-L1-expressing cells over low PD-L1-expressing cells; binding was further enhanced by interferon-γ, an inducer of PD-L1 expression. Binding affinities of PD-L1Pep-1 and PD-L1Pep-2 were approximately 373 and 281 nM, respectively. Cellular binding of the PD-L1-binding peptides was reduced by silencing PD-L1 gene expression or competition with anti-PD-L1 antibody. PD-L1Pep-1 and PD-L1Pep-2 induced the internalization and downregulated cell surface levels of PD-L1. The PD-L1-binding peptides restored cytokine secretion and T-cell proliferation to cells inhibited by co-culture with tumor cells or culture on PD-L1-coated plates. Intravenously injected PD-L1Pep-1 and PD-L1Pep-2 efficiently homed to tumor tissues, inhibited tumor growth, and increased CD8+/FoxP3+ ratio in mice. The PD-L1-binding peptides in combination with doxorubicin or PD-L1-targeted liposomal doxorubicin inhibited tumor growth and increased CD8+/FoxP3+ ratio more efficiently than doxorubicin alone and untargeted liposomal doxorubicin, respectively. These results suggest that PD-L1Pep-1 and PD-L1Pep-2 block PD-L1 and reinvigorate T-cell activity, inhibiting tumor growth by enhancing anti-tumor immunity.