Keita Un

Enhanced Transfection Efficiency into Macrophages and Dendritic Cells by a Combination Method Using Mannosylated Lipoplexes and Bubble Liposomes with Ultrasound Exposure

To achieve effective gene therapy, it is necessary to selectively and efficiently transfect therapeutic gene into targeted cells. In this study, we developed a combination method using mannosylated lipoplexes, which show selectivity to antigen-presenting cells such as macrophages and dendritic cells, and bubble liposomes (BLs), which are known to enhance transfection efficiency on exposure to ultrasound (US). In cultured mouse macrophages, known for the expression of mannose receptors, the transfection efficiency of this combination method using mannosylated lipoplexes and BLs with US was higher than that of naked pDNA or unmodified lipoplexes and BLs. In the liver and spleen, the in vivo transfection efficiency of this combination method was significantly higher than that of naked pDNA or unmodified lipoplexes and BLs with US. Transfection in hepatic nonparenchymal cells using this combination method was about 12 times higher than that in hepatic parenchymal cells. As far as splenic transfection activities were concerned, the transfection efficiency of this combination method in CD11c(+) cells was significantly higher than that in CD11c(-) cells. In conclusion, we demonstrated that the gene transfection efficiency in macrophages and dendritic cells was significantly increased by this combination method using mannosylated lipoplexes and BLs with US exposure.

Development of an ultrasound-responsive and mannose-modified gene carrier for DNA vaccine therapy.

Development of a gene delivery system to transfer the gene of interest selectively and efficiently into targeted cells is essential for achievement of sufficient therapeutic effects by gene therapy. Here, we succeeded in developing the gene transfection method using ultrasound (US)-responsive and mannose-modified gene carriers, named Man-PEG(2000) bubble lipoplexes. Compared with the conventional lipofection method using mannose-modified carriers, this transfection method using Man-PEG(2000) bubble lipoplexes and US exposure enabled approximately 500-800-fold higher gene expressions in the antigen presenting cells (APCs) selectively in vivo. This enhanced gene expression was contributed by the improvement of delivering efficiency of nucleic acids to the targeted organs, and by the increase of introducing efficiency of nucleic acids into the cytoplasm followed by US exposure. Moreover, high anti-tumor effects were demonstrated by applying this method to DNA vaccine therapy using ovalbumin (OVA)-expressing plasmid DNA (pDNA). This US-responsive and cell-specific gene delivery system can be widely applied to medical treatments such as vaccine therapy and anti-inflammation therapy, which its targeted cells are APCs, and our findings may help in establishing innovative methods for in-vivo gene delivery to overcome the poor introducing efficiency of carriers into cytoplasm which the major obstacle associated with gene delivery by non-viral carriers.

Suppression of Melanoma Growth and Metastasis by DNA Vaccination Using an Ultrasound-Responsive and Mannose-Modified Gene Carrier

DNA vaccination has attracted much attention as a promising therapy for the prevention of metastasis and relapse of malignant tumors, especially highly metastatic tumors such as melanoma. However, it is difficult to achieve a potent cancer vaccine effect by DNA vaccination, since the number of dendritic cells, which are the major targeted cells of DNA vaccination, is very few. Here, we developed a DNA vaccination for metastatic and relapsed melanoma by ultrasound (US)-responsive and antigen presenting cell (APC)-selective gene carriers reported previously, named Man-PEG₂₀₀₀ bubble lipoplexes. Following immunization using US exposure and Man-PEG(2000) bubble lipoplexes constructed with pUb-M, which expresses ubiquitylated melanoma-specific antigens (gp100 and TRP-2), the secretion of Th1 cytokines (IFN-γ and TNF-α) and the activities of cytotoxic T lymphocytes (CTLs) were specifically enhanced in the presence of B16BL6 melanoma antigens. Moreover, we succeeded in obtaining potent and sustained DNA vaccine effects against solid and metastatic tumor derived from B16BL6 melanoma specifically. The findings obtained from this study suggest that the gene transfection method using Man-PEG₂₀₀₀ bubble lipoplexes and US exposure could be suitable for DNA vaccination aimed at the prevention of metastatic and relapsed cancer.

Efficient suppression of murine intracellular adhesion molecule-1 using ultrasound-responsive and mannose-modified lipoplexes inhibits acute hepatic inflammation†

Hepatitis is often associated with the overexpression of various adhesion molecules. In particular, intracellular adhesion molecule‐1 (ICAM‐1), which is expressed on hepatic endothelial cells (HECs) in the early stage of inflammation, is involved in serious illnesses. Therefore, ICAM‐1 suppression in HECs enables the suppression of inflammatory responses. Here, we developed an ICAM‐1 small interfering RNA (siRNA) transfer method using ultrasound (US)‐responsive and mannose‐modified liposome/ICAM‐1 siRNA complexes (Man‐PEG2000 bubble lipoplexes [Man‐PEG2000 BLs]), and achieved efficient HEC‐selective ICAM‐1 siRNA delivery in combination with US exposure. Moreover, the sufficient ICAM‐1 suppression effects were obtained via this ICAM‐1 siRNA transfer in vitro and in vivo, and potent anti‐inflammatory effects were observed in various types of inflammation, such as lipopolysaccharide, dimethylnitrosamine, carbon tetrachloride, and ischemia/reperfusion‐induced inflammatory mouse models. Conclusion: HEC‐selective and efficient ICAM‐1 siRNA delivery using Man‐PEG2000 BLs and US exposure enables suppression of various types of acute hepatic inflammation. This novel siRNA delivery method may offer a valuable system for medical treatment where the targeted cells are HECs. (HEPATOLOGY 2012;56:259–269)

The elucidation of gene transferring mechanism by ultrasound-responsive unmodified and mannose-modified lipoplexes.

The development of gene transfection methods enhancing the level of gene expression under simple and low-toxic condition is required for gene therapy in clinical. Our group has developed the ultrasound (US)-mediated gene transfection method using Man-PEG(2000) bubble lipoplexes, which are US-responsive and mannose-modified gene carriers, and succeeded in obtaining the enhanced gene expression in mannose receptor-expressing cells selectively by the gene transfer using Man-PEG(2000) bubble lipoplexes with US exposure in vitro and in vivo. Here, we investigated pDNA transferring mechanism followed by US exposure to unmodified and Man-PEG(2000) bubble lipoplexes, in particular, focused on US exposure timing. Following investigation of intracellular transferring characteristics, a large amount of pDNA was transferred into the cytoplasm followed by US-mediated destruction of bubble lipoplexes in the gene transfer using both bubble lipoplexes with US exposure. Moreover, the effective gene expression was obtained without TNF-α production when US was exposed until 5 min after the addition of bubble lipoplexes. These findings suggest that the gene transfer using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure enables to transfer pDNA into the cytoplasm, and optimized US exposure timing is important to achieve the high level of gene expression and the low level of pro-inflammatory cytokine production.

Involvement of activated transcriptional process in efficient gene transfection using unmodified and mannose-modified bubble lipoplexes with ultrasound exposure

Recently, our group developed ultrasound (US)-responsive and mannose-modified gene carriers (Man-PEG(2000) bubble lipoplexes), and successfully obtained a high level of gene expression in mannose receptor-expressing cells following gene transfection using Man-PEG(2000) bubble lipoplexes and US exposure. We also reported that large amounts of plasmid DNA (pDNA) were transferred into the cytoplasm of the targeted cells in the gene transfection using this method. In the present study, we investigated the involvement of transcriptional processes on enhanced gene expression obtained by unmodified and Man-PEG(2000) bubble lipoplexes with US exposure. The transcriptional process related to activator protein-1 (AP-1) and nuclear factor-κB (NFκB) was activated by US exposure, and was founded to be involved in enhanced gene expression obtained by gene transfection using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure. On the other hand, activation of AP-1 and NFκB pathways followed by US exposure was hardly involved in the inflammatory responses in the gene transfection using this method. These findings suggest that activation of AP-1 and NFκB followed by US exposure is involved in the enhanced gene expression using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure, and the selection of pDNAs activated by US exposure is important in this gene transfection method.

Synthesis and evaluation of glyco-coated liposomes as drug carriers for active targeting in drug delivery systems

Novel sugar-conjugated cholesterols, β-Gal-, α-Man-, β-Man-, α-Fuc-, and β-Man-6P-S-β-Ala-Chol, were synthesized and incorporated into liposomes. In vitro experiments using the glyco-coated liposomes showed that the glyco-coated liposomes are efficiently taken up by cells expressing carbohydrate-binding receptors selectively. Glyco-coated liposomes are promising candidates for drug delivery vehicles.

Enhancement of the anti-tumor effect of DNA vaccination using an ultrasound-responsive mannose-modified gene carrier in combination with doxorubicin-encapsulated PEGylated liposomes.

A method involving the use of doxorubicin-loaded polyethylene-glycol-modified liposomes and transfection using mannose-modified bubble lipoplexes in combination with ultrasound irradiation may be a promising approach to cancer treatment; it could not only suppress early-stage tumor growth but also enhance transfection efficacy in antigen-presenting cells, thus enhancing the therapeutic potential of a DNA vaccine. However, to date only limited research has been carried out regarding this combination DNA vaccination method for use in cancer therapy. In this study, we examined the anti-tumor effect of DNA vaccination using an ultrasound-responsive mannose-modified gene carrier combined with doxorubicin-encapsulated polyethylene-glycol-modified liposomes. Doxorubicin-encapsulated PEGylated liposomes activated transcriptional factors, such as nuclear factor-κB and AP-1 in the spleen; subsequently pUb-M, ubiquitylated melanoma-related antigen encoding plasmid DNA expression in splenic cells was significantly enhanced. Moreover, effective cytotoxic T-lymphocyte activities were stimulated by DNA vaccination combined with the administration of doxorubicin-encapsulated polyethylene-glycol-modified liposomes. Furthermore, potent DNA vaccine effects against established solid tumor and metastatic tumor derived from B16BL6 melanoma were observed. These results suggest that the combined use of DNA vaccination with doxorubicin-encapsulated polyethylene-glycol-modified liposomes could be an effective method for the treatment of melanoma using immunotherapy.

Evaluation of inflammatory responses due to small interfering RNA transfer using unmodified- and mannose-modified bubble lipoplexes with ultrasound exposure in primary cultured macrophages

Abstract Development of an efficient small interfering RNA (siRNA) delivery method using non-viral carriers is necessary to determine potent therapeutic effects of RNA interference. Inflammatory responses induced by siRNA interaction with Toll-like receptors and retinoic-acid-inducible gene I protein/melanoma differentiation-associated gene 5 (RIG-I/MDA-5) are obstacles to the application of siRNAs in clinically. Here, we evaluated the effects on inflammatory responses by our siRNA delivery method using bubble lipoplexes with ultrasound (US) exposure in cultured macrophages. The effective gene suppression effects were obtained under low-toxic conditions in this siRNA transfer method. The interferon (IFN)-α after siRNA transfer using lipoplexes/bubble lipoplexes with US exposure was not detected. However, low levels of type I IFN mRNA production were induced through interaction of siRNA and cytoplasmic RIG-I/MDA-5, but not Toll-like receptors. Our findings indicate that it is possible to develop a safe and efficient siRNA delivery technique using mannosylated bubble lipoplexes and US exposure.