New strategy for MS treatment with autoantigen-modified liposomes and their therapeutic effect.

As current treatments for multiple sclerosis (MS) remain chemotherapeutic ones directed toward symptoms, the development of a curative treatment is urgently required. Herein, we show an autoreactive immune cell-targetable approach using autoantigen-modified liposomes for the curative treatment of MS. In these experiments, experimental autoimmune encephalomyelitis (EAE) induced by autoantigenic myelin oligodendrocyte glycoprotein (MOG) peptide was used as a model of primary progressive MS, and MOG-modified liposomes encapsulating doxorubicin (MOG-LipDOX) were used as a therapeutic drug. The results showed that the progression of encephalomyelitis symptoms was significantly suppressed by MOG-LipDOX injection, whereas the other samples failed to show any effect. Additionally, invasion of inflammatory immune cells into the spinal cord and demyelination of neurons were clearly suppressed in the MOG-LipDOX-treated mice. FACS analysis revealed that the number of both MOG-recognizable CD4+ T cells in the spleen was obviously decreased after MOG-LipDOX treatment. Furthermore, the number of effector Th17 cells in the spleen was significantly decreased and that of regulatory Treg cells was concomitantly increased. Finally, we demonstrated that myelin proteolipid protein (PLP)-modified liposomes encapsulating DOX (PLP-LipDOX) also showed the therapeutic effect on relapsing-remitting EAE. These findings indicate that autoantigen-modified liposomal drug produced a highly therapeutic effect on EAE by delivering the encapsulated drug to autoantigen-recognizable CD4+ T cells and thus suppressing autoreactive immune responses. The present study suggests that the use of these autoantigen-modified liposomes promises to be a suitable therapeutic approach for the cure of MS.

Development of tissue factor-targeted liposomes for effective drug delivery to stroma-rich tumors.

Tissue factor (TF), which is well known as a trigger molecule of extrinsic coagulation, is found in not only tumor cells but also in stromal cells in tumor tissues. Thus, TF is a candidate molecule to potentially enable targeting of both tumor cells and stromal cells for anti-cancer drug delivery. Herein, we prepared liposomes conjugated with the Fab' fragment of anti-TF antibody (TF Ab-Lip) and evaluated the capability for drug delivery to stroma-rich tumors for realizing a whole tumor tissue-targetable strategy. When the targetability of TF Ab-Lip to TF-expressing KLN205 squamous tumor cells and NIH3T3 fibroblast cells were examined, TF Ab-Lip was significantly taken up into both cells compared with non-targeted liposomes. Corresponding to this result, doxorubicin-encapsulated TF Ab-Lip (TF Ab-LipDOX) showed potent cytotoxicity against KLN205 cells. In vivo experiments using KLN205 solid tumor-bearing mice indicated that TF Ab-Lip became highly accumulated and distributed widely in not only the tumor cell region but also in the stromal one in the tumor. Treatment with TF Ab-LipDOX significantly suppressed the growth of KLN205 solid tumors. Furthermore, TF Ab-Lip targetable both mouse and human TF (mhTF Ab-Lip) became distributed throughout stroma-rich human pancreatic BxPC3 tumors and the treatment of the BxPC3 tumor-bearing mice with mhTF Ab-LipDOX showed highest tumor-suppressive effect. These data suggest that TF Ab-Lip could achieve effective accumulation for stroma-rich tumor treatment.

Macrophage-targeted, enzyme-triggered fluorescence switch-on system for detection of embolism-vulnerable atherosclerotic plaques.

The development of atherosclerotic plaques is a critical step that can result in an arterial embolism. Therefore, detection of these vulnerable plaques is of clinical significance for the diagnosis of atherosclerosis. However, there are few imaging systems able to detect such plaques easily. In this study, we designed a new platform for near-infrared fluorescence (NIRF) imaging of macrophages in atherosclerotic plaques, one using both a liposomal DDS and an activatable fluorescent probe, and evaluated the utility of this imaging for the diagnosis of atherosclerosis. We first synthesized a fluorescent switch-on probe, Peptide-ICG2, which is optically silent under normal conditions but activated in the presence of the lysosomal enzyme, cathepsin B. To achieve macrophage-specific fluorescence activation, we encapsulated Peptide-ICG2 into phosphatidylserine-containing liposome (P-ICG2-PS-Lip), since the accumulation of phosphatidylserine receptor-bearing macrophages is characteristic of embolism-vulnerable plaques. The experiments using macrophage-like RAW264 cells in culture showed that P-ICG2-PS-Lip was selectively taken up into the cells and that significant fluorescence of the probe was observed. For NIRF imaging of the atherosclerotic plaques, P-ICG2-PS-Lip was intravenously injected into ApoE-knockout atherosclerotic model mice or WHHL rabbits, and the fluorescence at the aortae was imaged. The results indicated that ICG fluorescence could be successfully observed at the plaques on the artery walls. The results of the present study thus suggest that NIRF imaging using P-ICG2-PS-Lip would be useful for detecting embolism-vulnerable atherosclerotic plaques.

A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165).

Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.