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

Novel sugar-conjugated cholesterols, ß-Gal-, α-Man-, ß-Man-, α-Fuc-, and ß-Man-6P-S-ß-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.

Determinants for in vivo antitumor effect of angiogenesis inhibitor SU5416 formulated in PEGylated emulsion.

Angiogenesis, the sprouting of capillaries from preexisting ones, is essential for the sustained growth of solid tumors. In this study, we used SU5416, a hydrophobic molecule with potent tyrosine kinase inhibitor of type 2 receptor for vascular endothelial growth factor (VEGF), as PEGylated emulsion (SU5416-PE), and evaluated the antitumor potency of this formulation in Lewis lung cancer (LLC), Colon-26 (C26), and B16BL6 melanoma (B16) tumor-bearing mice. Intravenous injection of SU5416-PE into tumor-bearing mice significantly suppressed the growth of C26 and B16 tumors, but had no effect on the growth of LLC tumors. MTT assay revealed that SU5416 inhibited the proliferation of human umbilical vein endothelial cells in a concentration-dependent manner but did not show such an inhibitory effect on all types of tumor cells examined, demonstrating the specificity of SU5416 for endothelial cells. Considering that VEGF levels within C26 and B16 tumors were found to be about 10-fold and 20-fold higher than that in LLC tumors, respectively, it was suggested that SU5416-PE would inhibit angiogenesis in certain types of tumor tissue such as C26 and B16 where VEGF plays a major role for promoting angiogenesis, leading to the suppression of in vivo tumor growth.

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

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.

Nanoparticle-based passive drug targeting to tumors: considerations and implications for optimization.

There are many potential barriers to the effective delivery of small-molecule drugs to solid tumors. Most small-molecule chemotherapeutic drugs have a large volume of distribution upon intravenous administration, which is often associated with a narrow therapeutic index due to their high level of toxicity in healthy tissues. Nanoparticle-based therapeutics for tumor targeting have emerged as one of the promising approaches to overcome the lack of tissue specificity of conventional chemotherapeutic drugs. Various different concepts have been envisioned for nanoparticle-mediated drug targeting. Among them, the passive drug targeting strategy has been the most widely investigated, and numerous preclinical studies have provided insights into the validity of the strategy. This review article briefly introduces our recent findings related to the passive drug targeting strategy including its application in anti-angiogenic therapy, along with considerations to be taken into account and implications for the rational design of a passive drug targeting strategy.

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.

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.

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.

In vivo anti-tumor effect of PEG liposomal doxorubicin (DOX) in DOX-resistant tumor-bearing mice: Involvement of cytotoxic effect on vascular endothelial cells.

We evaluated the in vivo anti-tumor effect of polyethylene glycol-modified liposomal doxorubicin (PEG liposomal DOX) in the DOX-resistant Colon-26 cancer cells (C26/DOX)-bearing mice model. IC(50) value of DOX to C26/DOX in vitro (40.0 microM) was about 250 times higher than that to control C26 (C26/control) (0.15 microM). However, in vivo anti-tumor effect of PEG liposomal DOX was similar in both C26/control- and C26/DOX-bearing mice, suggesting that the in vivo anti-tumor effect of PEG liposomal DOX was not directly reflecting the sensitivity of these tumor cells to DOX. IC(50) value (0.10 microM) of DOX to HUVEC, a model vascular endothelial cell, was similar to that of C26/control. Double immunohistochemical staining of vascular endothelial cells and apoptotic cells within the tumor tissue after intravenous administration of PEG liposomal DOX showed that the extent of co-localization of apoptotic cells with endothelial cells was significantly higher for C26/DOX tumors (60%) than C26/control ones (20%), suggesting that the apoptosis is caused preferentially for vascular endothelial cells in C26/DOX tumor. From these results, it was suggested that the cytotoxic effect of DOX on vascular endothelial cells in the tumor would be involved in the in vivo anti-tumor effect of PEG liposomal DOX in C26/DOX-bearing mice.

Determinants for in vivo anti-tumor effects of PEG liposomal doxorubicin: importance of vascular permeability within tumors.

To elucidate the determinants of the in vivo anti-tumor efficacy of polyethylene glycol (PEG)-modified liposomal doxorubicin (DOX), we examined its anti-tumor effect against three different tumor cell lines (Lewis lung cancer (LLC), Colon-26 (C26) and B16BL6 melanoma (B16)) in vitro and in vivo. In vitro, LLC was the most sensitive tumor to DOX and liposomal DOX based on the MTT assay. However, the strongest in vivo anti-tumor effect was observed in the C26 tumor-bearing mice. The in vivo accumulation of radiolabelled PEG liposome in the C26 tumor after intravenous injection was significantly larger than in other tumors. The extent of vascularity assessed by immunohistochemical staining of CD31 was not directly related with the tumor accumulation of PEG liposome. On the other hand, Evans blue extravasation and secretion of VEGF in C26 tumors were higher than in LLC tumors, clearly demonstrating that the vasculature permeability was higher within C26 tumors. These results indicated that the vascular permeability within the tumor substantially affects the tumor accumulation of PEG liposome and may be one of the important determinants in the in vivo anti-tumor efficacy of PEG liposomal DOX.