Yoko Endo-Takahashi

Preparation of Angiopep-2 Peptide-Modified Bubble Liposomes for Delivery to the Brain

In the development of therapeutic approaches for central nervous system diseases, a significant obstacle is efficient drug delivery across the blood-brain barrier owing to its low permeability. Various nanocarriers have been developed for brain-targeted drug delivery by modification with specific ligands. We have previously developed polyethylene glycol-modified liposomes (Bubble liposomes [BLs]) that entrap ultrasound (US) contrast gas and can serve as both plasmid DNA or small interfering RNA carriers and US contrast agents. In this study, we attempted to prepare brain-targeting BLs modified with Angiopep-2 (Ang2) peptide (Ang2-BLs). Ang2 is expected to be a useful ligand for the efficient delivery of nanocarriers to the brain. We showed that Ang2-BLs interacted specifically with brain endothelial cells via low-density lipoprotein receptor-related protein-1. We also confirmed that Ang2-BLs could entrap US contrast gas and had US imaging ability as well as unmodified BLs. Furthermore, we demonstrated that Ang2-BLs accumulated in brain tissue after intravascular injection. These results suggested that Ang2-BLs may be a useful tool for brain-targeted delivery and US imaging via systemic administration.

Systemic delivery of miR-126 by miRNA-loaded Bubble liposomes for the treatment of hindlimb ischemia

Currently, micro RNA (miRNA) is considered an attractive target for therapeutic intervention. A significant obstacle to the miRNA-based treatments is the efficient delivery of miRNA to the target tissue. We have developed polyethylene glycol-modified liposomes (Bubble liposomes (BLs)) that entrap ultrasound (US) contrast gas and can serve as both plasmid DNA (pDNA) or small interfering RNA (siRNA) carriers and US contrast agents. In this study, we investigated the usability of miRNA-loaded BLs (mi-BLs) using a hindlimb ischemia model and miR-126. It has been reported that miR-126 promotes angiogenesis via the inhibition of negative regulators of VEGF signaling. We demonstrated that mi-BLs could be detected using diagnostic US and that mi-BLs with therapeutic US could deliver miR-126 to an ischemic hindlimb, leading to the induction of angiogenic factors and the improvement of blood flow. These results suggest that combining mi-BLs with US may be useful for US imaging and miRNA delivery.

Efficient siRNA delivery using novel siRNA-loaded Bubble liposomes and ultrasound

Recently, we developed novel polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) entrapping an ultrasound (US) imaging gas and reported that the combination of BLs and US was useful for the delivery of siRNA directly into the cytoplasm. However, the results were obtained using a mixture of BLs and naked siRNA. With systemic injections, it is important to control the biodistribution of both BLs and siRNA. In addition, the delivery of siRNA is affected by nuclease degradation after intravenous administration. In this study, we prepared novel siRNA-loaded BLs (si-BLs) using a cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). We demonstrated that siRNA could be loaded onto BLs containing DOTAP and that siRNA-loaded BLs were stable in serum. A specific gene-silencing effect was also achieved by transfection with si-BLs. Thus, the combination of si-BLs with US exposure can be used for delivery of siRNA to a specific tissue via systemic injection.

Delivery of an Angiogenic Gene into Ischemic Muscle by Novel Bubble Liposomes Followed by Ultrasound Exposure

ABSTRACTPurposeTo develop a safe and efficient gene delivery system into skeletal muscle using the combination of Bubble liposomes (BL) and ultrasound (US) exposure, and to assess the feasibility and the effectiveness of BL for angiogenic gene delivery in clinical use.MethodsA solution of luciferase-expressing plasmid DNA (pDNA) and BL was injected into the tibialis (TA) muscle, and US was immediately applied to the injection site. The transfection efficiency was estimated by a luciferase assay. The ischemic hindlimb was also treated with BL and US-mediated intramuscular gene transfer of bFGF-expressing plasmid DNA. Capillary vessels were assessed using immunostaining. The blood flow was determined using a laser Doppler blood flow meter.ResultsHighly efficient gene transfer could be achieved in the muscle transfected with BLs, and US mediated the gene transfer. Capillary vessels were enhanced in the treatment groups with this gene transfer method. The blood flow in the treated groups with this gene transfer method quickly recovered compared to other treatment groups (non-treated, bFGF alone, or bFGF+US).ConclusionThe gene transfer system into skeletal muscle using the combination of BL and US exposure could be an effective means for angiogenic gene therapy in limb ischemia.

CXCL17 Expression by Tumor Cells Recruits CD11b+Gr1highF4/80- Cells and Promotes Tumor Progression

Background Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1), recruits immature myeloid-derived cells and enhances early tumor progression. Methodology/Principal Findings CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b+Gr1+ myeloid-derived cells at tumor sites in mice and promoted CD31+ tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b+Gr1highF4/80− cells (∼90%) with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b+Gr1+ cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation. Conclusions/Significance These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis.

pDNA-loaded Bubble liposomes as potential ultrasound imaging and gene delivery agents

We have developed polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) that entrap ultrasound (US) contrast gas, and we have reported that the combination of BLs and US exposure was an effective tool for delivering pDNA and siRNA in vitro and in vivo. In this study, we prepared pDNA-loaded BLs using three types of cationic lipids to enhance the US imaging effect and the transfection efficiency via systemic injection. We investigated the US imaging abilities of these BLs, their protective effects on pDNA from serum component, and their transfection effects in vitro and in vivo. As a result, we demonstrated that the US imaging ability and transfection effect varied with lipid component and that p-BLs containing DSDAP could be the most stable and effective tool the among three types of p-BLs. Indeed, in ischemic muscle, p-BLs containing DSDAP could be detected using diagnostic US and could deliver bFGF-expressing pDNA using therapeutic US, leading to the induction of angiogenic factors and the improvement of blood flow. These results suggest that combining p-BLs with US exposure may be useful for stable US imaging and efficient gene delivery and may lead to the establishment of a theranostic approach, which is a combination of disease diagnosis and therapy.

Systemic delivery systems of angiogenic gene by novel bubble liposomes containing cationic lipid and ultrasound exposure.

Recently, we developed polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) entrapping ultrasound (US) gas and reported that the combination of BL and US exposure was an effective tool for the delivery of pDNA directly into skeletal muscles of an ischemic hindlimb model with local injection. To achieve gene delivery to deeper tissues, we attempted to prepare novel Bubble liposomes which were able to be loaded with pDNA and useful for systemic injection. We prepared BLs using cationic lipid and analyzed the interaction with the BLs and pDNA using flow cytometry. The solution of pDNA-loaded BLs (p-BLs) was further injected into the tail vein of hindlimb ischemia model mice, and transdermal US exposure was applied to ischemic hindlimb. The effects of transfection on angiogenic factors were investigated by real-time PCR. Blood flow was determined using a laser Doppler blood flow meter. The interaction with BLs and pDNA increased in the presence of DOTAP and short PEG chains and resulted in increased stability of pDNA in the serum. Transfection with pDNA encoding the bFGF gene using p-BLs and US induced various angiogenic factors and improved the blood flow. The gene delivery system into the ischemic hindlimb using the combination of p-BLs and US exposure could be an effective tool for angiogenic gene therapy via systemic injection.

Bubble liposomes and ultrasound promoted endosomal escape of TAT-PEG liposomes as gene delivery carriers.

We have previously developed laminin-derived AG73 peptide-labeled poly(ethylene glycol)-modified liposomes (AG73-PEG liposomes) for selective cancer gene therapy and reported that Bubble liposomes (BLs) and ultrasound (US) exposure could accelerate the endosomal escape of AG73-PEG liposomes, leading to the enhancement of transfection efficiency; however, it is still unclear whether BLs and US exposure can also enhance the transfection efficiency of other vectors. We therefore assessed the effect of BLs and US exposure on the gene transfection efficiency of trans-activating transcriptor (TAT) peptide modified PEG liposomes. Although TAT-PEG liposomes were efficiently internalized into cells, the efficacy of endosomal escape was insufficient. The transfection efficiencies of TAT-PEG liposomes were enhanced by about 30-fold when BLs and US exposure were used. We also confirmed that BLs and US exposure could not enhance the direct transportation of TAT-PEG liposomes into cells. Confocal microscopy showed that BLs and US exposure promoted endosomal escape of TAT-PEG liposomes. Our results suggested that BLs and US exposure could enhance transfection efficiency by promoting endosomal escape, which was independent of modified molecules of carriers. Thus, BLs and US exposure can be a useful tool to achieve efficient gene transfection by improving endosomal escape of various carriers.

Enhanced gene delivery using Bubble liposomes and ultrasound for folate-PEG liposomes

We have previously reported that the transfection efficiency of laminin-derived AG73-peptide labeled polyethyleneglycol-modified liposomes (AG73-PEG liposomes) was enhanced by echo-contrast gas entrapping PEG liposomes (Bubble liposomes, BLs) and ultrasound (US) exposure by improving endosomal escape. However, it has not been well understood whether BLs and US exposure can enhance the transfection efficiency of other carriers except AG73-PEG liposomes. In this study, to evaluate whether BLs and US exposure can be generally applied to gene delivery carriers, we focused on folate as a model ligand and examined whether BLs and US exposure could enhance the transfection efficiency of folate-PEG liposomes. Folate-PEG liposomes could internalize into cells efficiently, whereas they could not deliver genes into cytosol from endosomes sufficiently. BLs and US exposure could enhance the transfection efficiency of folate-PEG liposomes compared with folate-PEG liposomes alone without their direct induction into cells. These results suggested that BLs and US exposure could enhance the transfection efficiency of folate-PEG liposomes in the same manner as AG73-PEG liposomes. Thus, BLs and US exposure may be a promising tool to achieve efficient gene transfection into various gene carriers in general.

Local Gene Delivery System by Bubble Liposomes and Ultrasound Exposure into Joint Synovium

Recently, we have developed novel polyethylene glycol modified liposomes (bubble liposomes; BL) entrapping an ultrasound (US) imaging gas, which can work as a gene delivery tool with US exposure. In this study, we investigated the usefulness of US-mediated gene transfer systems with BL into synoviocytes in vitro and joint synovium in vivo. Highly efficient gene transfer could be achieved in the cultured primary synoviocytes transfected with the combination of BL and US exposure, compared to treatment with plasmid DNA (pDNA) alone, pDNA plus BL, or pDNA plus US. When BL was injected into the knee joints of mice, and US exposure was applied transcutaneously to the injection site, highly efficient gene expression could be observed in the knee joint transfected with the combination of BL and US exposure, compared to treatment with pDNA alone, pDNA plus BL, or pDNA plus US. The localized and prolonged gene expression was also shown by an in vivo luciferase imaging system. Thus, this local gene delivery system into joint synovium using the combination of BL and US exposure may be an effective means for gene therapy in joint disorders.