Daiki Omata

Delivery of siRNA into the cytoplasm by liposomal bubbles and ultrasound

Small interfering RNA (siRNA) is expected to be a novel therapeutic tool, however, its utilization has been limited by inefficient delivery systems. Recently, we have developed novel polyethyleneglycol 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 whether the BL were suitable for the delivery of siRNA. BL efficiently delivered siRNA with only 10 s of exposure to US in vitro. Specific gene silencing effects could be achieved well even in the presence of serum or with the disruption of endocytosis. We suggest that siRNA is directly introduced into the cytoplasm by the BL and US and the mechanism enables effective transfection within a short time and in the presence of high serum. Transfection of siRNA into the tibialis muscles with BL and US was also performed. The gene-silencing effect could be sustained for more than 3 weeks. Thus, BL could be a useful siRNA delivery tool in vitro and in vivo.

Enhanced Laminin-Derived Peptide AG73-Mediated Liposomal Gene Transfer by Bubble Liposomes and Ultrasound

A promising strategy as a cancer therapeutic is tumor-targeted gene delivery. The AG73 peptide derived from the laminin alpha1 chain is a ligand for syndecans, and syndecan-2 is highly expressed in some cancer cells. In this study, AG73-PEG liposomes were developed for selective gene delivery to syndecan-2 overexpressing cancer cells. AG73-PEG liposomes were used in combination with Bubble liposomes and ultrasound exposure to enhance transfection efficiency by promoting the escape of the liposomes from the endosome to the cytosol. AG73-PEG liposomes showed selective gene delivery to syndecan-2 overexpressing cancer cells. Furthermore, AG73-mediated liposomal gene transfection efficiency was enhanced by 60-fold when Bubble liposomes and ultrasound exposure were used, despite the absence of an increase in the uptake of AG73-PEG liposomes into the cells. Confocal microscope analysis revealed that the Bubble liposomes and ultrasound promoted intracellular trafficking of the AG73-PEG liposomes during gene transfection. Thus, the combination of AG73-PEG liposomes with Bubble liposomes and ultrasound exposure may be a promising method to achieve selective and efficient gene delivery for cancer therapy.

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.

Cancer cell specific gene delivery by laminin-derived peptide AG73-labeled liposomes

We developed laminin-derived AG73 peptide labeled liposomes for cancer specific gene therapy. AG73 peptide is well known as a ligand for syndecan-2 which is highly expressed in various cancer cells. Liposomes labeled with AG73 showed high efficient transfection efficiency in syndecan-2 overexpressing cells, and found that AG73 could be a superior molecule in the development of non-viral vector using liposomes for the gene delivery to syndecan-2 overexpressing cancer cells.

Tumor growth suppression by the combination of nanobubbles and ultrasound.

We previously developed novel liposomal nanobubbles (Bubble liposomes [BL]) that oscillate and collapse in an ultrasound field, generating heat and shock waves. We aimed to investigate the feasibility of cancer therapy using the combination of BL and ultrasound. In addition, we investigated the anti‐tumor mechanism of this cancer therapy. Colon‐26 cells were inoculated into the flank of BALB/c mice to induce tumors. After 8 days, BL or saline was intratumorally injected, followed by transdermal ultrasound exposure of tumor tissue (1 MHz, 0–4 W/cm2, 2 min). The anti‐tumor effects were evaluated by histology (necrosis) and tumor growth. In vivo cell depletion assays were performed to identify the immune cells responsible for anti‐tumor effects. Tumor temperatures were significantly higher when treated with BL + ultrasound than ultrasound alone. Intratumoral BL caused extensive tissue necrosis at 3–4 W/cm2 of ultrasound exposure. In addition, BL + ultrasound significantly suppressed tumor growth at 2–4 W/cm2. In vivo depletion of CD8+ T cells (not NK or CD4+ T cells) completely blocked the effect of BL + ultrasound on tumor growth. These data suggest that CD8+ T cells play a critical role in tumor growth suppression. Finally, we concluded that BL + ultrasound, which can prime the anti‐tumor cellular immune system, may be an effective hyperthermia strategy for cancer treatment.

Nonviral Gene Delivery Systems by the Combination of Bubble Liposomes and Ultrasound

The combination of therapeutic ultrasound (US) and nano/microbubbles is an important system for establishing a novel and noninvasive gene delivery system. Genes are delivered more efficiently using this system compared with a conventional nonviral vector system such as the lipofection method, resulting in higher gene expression. This higher efficiency is due to the gene being delivered into the cytosol and bypassing the endocytosis pathway. Many in vivo studies have demonstrated US-mediated gene delivery with nano/microbubbles, and several gene therapy feasibility studies for various diseases have been reported. In addition, nano/microbubbles can deliver genes site specifically by the control of US exposure site. In the present review, we summarize the gene delivery systems by the combination of nano/microbubbles and US, describe their properties, and assess applications and challenges of US theranostics.

Effects of doxorubicin-encapsulating AG73 peptide-modified liposomes on tumor selectivity and cytotoxicity

Doxorubicin-encapsulating liposomal formulations, known as Doxil, have been used for the treatment of Kaposis sarcoma and ovarian cancer. However, there is still a need for a drug delivery system for doxorubicin that limits the treatments side effects, namely, mucositis and hand-and-foot syndrome. The AG73 peptide derived from the laminin α1 chain is a ligand for syndecans, and syndecan-2 is highly expressed in some cancer cells. In this study, to develop a safer and more selective liposomal formulation, we prepared doxorubicin-encapsulating AG73 peptide-modified liposomes (AG73-Dox). First, we assessed the selectivity of AG73-Dox for cancer cells, including syndecan-2 over-expressing cells, using flow cytometry and confocal microscopy. AG73-Dox showed selective cellular uptake on cancer cells and enhancement of the intracellular uptake. Next, we examined the cytotoxicity of AG73-Dox using a WST assay. AG73-Dox exhibited a higher cytotoxicity against cancer cells than other control liposomes. In addition, we showed that the antitumor efficacy of AG73-Dox in vivo was better than that of free Dox. When we examined the biodistribution of liposomes, AG73 peptide-modified liposomes (AG73-L) tended to bind to intratumoral vessels and extravasated in the tumor tissue. Thus, further optimization of AG73-L toward tumor targeting may lead to a development of a useful tool for cancer therapy.

Ultrasound‐targeted Bubble Liposome Destruction Enhances AG73‐mediated Gene Transfer by Improvement of Intracellular Trafficking

For selective cancer gene therapy, we have developed AG73‐labeled polyethyleneglycol‐modified liposomes (AG73‐PEG liposomes) capable of encapsulating a gene condensed by poly‐l‐lysine. The present study examined whether echo‐contrast gas‐entrapping liposomes, also known as Bubble liposomes (BLs), and ultrasound (US) exposure affect not only the cell membrane but also intracellular vesicles, and enhance the release of pDNA from endosomes into the cytoplasm to achieve efficient gene transfection. AG73‐mediated liposomal gene transfection efficiency was enhanced when BLs and US exposure were used. Furthermore, confocal microscopic analysis revealed that the BLs and US exposure promoted intracellular trafficking of the AG73‐PEG liposomes during gene transfection. Thus, the use of AG73‐PEG liposomes together with BLs and US exposure may be a promising way to achieve selective and efficient gene delivery.