Yu Sakurai

A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo.

Modification of liposomal siRNA carriers with polyethylene glycol, i.e., PEGylation, is a generally accepted strategy for achieving in vivo stability and delivery to tumor tissue. However, PEGylation significantly inhibits both cellular uptake and the endosomal escape process of the carriers. In a previous study, we reported on the development of a multifunctional envelope-type nano device (MEND) for siRNA delivery and peptide-based functional devices for overcoming the limitations and succeeded in the efficient delivery of siRNA to tumors. In this study, we synthesized a pH-sensitive cationic lipid, YSK05, to overcome the limitations. The YSK05-MEND had a higher ability for endosomal escape than other MENDs containing conventional cationic lipids. The PEGylated YSK05-MEND induced efficient gene silencing and overcame the limitations followed by optimization of the lipid composition. Furthermore, the intratumoral administration of the YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids. Collectively, these data confirm that YSK05 facilitates the endosomal escape of the MEND and thereby enhances the efficacy of siRNA delivery into cytosol and gene silencing.

Endosomal escape and the knockdown efficiency of liposomal-siRNA by the fusogenic peptide shGALA

An siRNA that specifically silences the expression of mRNA is a potential therapeutic agent for dealing with many diseases including cancer. However, the poor cellular uptake and bioavailability of siRNA remains a major obstacle to clinical development. For efficient delivery to tumor tissue, the pharmacokinetics and intracellular trafficking of siRNA must be rigorously controlled. To address this issue, we developed a liposomal siRNA carrier, a multi-functional nano device (MEND). We describe herein an approach for systemic siRNA delivery to tumors by combining the MEND system with shGALA, a fusogenic peptide. In cultured cell experiments, shGALA-modification enhanced the endosomal escape of siRNA encapsulated in a polyethylene glycol modified MEND (PEG-MEND), resulting in an 82% knockdown of the target gene. In vivo systemic administration clarified that the shGALA-modified MEND (shGALA-MEND) showed 58% gene silencing in tumor tissues at a dose of 4 mg of siRNA/kg body weight. In addition, a significant inhibition of tumor growth was observed only for the shGALA-MEND and no somatic or hepatic toxicity was observed. Given the above data, this peptide-modified delivery system, a shGALA-MEND has great potential for the systemic delivery of therapeutic siRNA aimed at cancer therapy.

Hepatic Monoacylglycerol O-acyltransferase 1 as a Promising Therapeutic Target for Steatosis, Obesity, and Type 2 Diabetes

Over the past decade, considerable advances have been made in the discovery of gene targets in metabolic diseases. However, in vivo studies based on molecular biological technologies such as the generation of knockout mice and the construction of short hairpin RNA vectors require considerable effort and time, which is a major limitation for in vivo functional analysis. Here, we introduce a liver-specific nonviral small interfering RNA (siRNA) delivery system into rapid and efficient characterization of hepatic gene targets in metabolic disease mice. The comparative transcriptome analysis in liver between KKAy diabetic and normal control mice demonstrated that the expression of monoacylglycerol O-acyltransferase 1 (Mogat1), an enzyme involved in triglyceride synthesis and storage, was highly elevated during the disease progression. The upregulation of Mogat1 expression in liver was also found in other genetic (db/db) and diet-induced obese mice. The silencing of hepatic Mogat1 via a liver-specific siRNA delivery system resulted in a dramatic improvement in blood glucose levels and hepatic steatosis as well as overweight with no apparent overall toxicities, indicating that hepatic Mogat1 is a promising therapeutic target for metabolic diseases. The integrated approach with transcriptomics and nonviral siRNA delivery system provides a blueprint for rapid drug discovery and development.

Heterogeneity of tumor endothelial cells and drug delivery

To date anti-angiogenic therapy has been used for cancer therapy widely, yielding promising results. However, it has been elucidated that current anti-angiogenic drug has several issues to be solved, such as side-effects and drug resistance. It has been reported that tumor endothelial cells (TECs) differ from normal counterparts. In addition, it was shown that the TECs are heterogeneous according to the malignancy status of tumor. The development of novel strategy for targeting tumor vasculature is required. Recently, we have developed an active targeting system, which targets TECs specifically. In this review, we will discuss how TECs in tumor vasculature are heterogeneous and offer new perspectives on a drug delivery system, which can target heterogeneous tumor blood vessels from a viewpoint of personalized medicine.

Improvement of doxorubicin efficacy using liposomal anti-polo-like kinase 1 siRNA in human renal cell carcinomas.

It is well-known that renal cell carcinomas (RCCs) are resistant to classical cytotoxic anticancer drugs. Therefore, facilitating the impact of anticancer drugs by altering the cell phenotype should be a useful strategy for circumventing this. We developed a multifunctional envelope-type nanodevice (MEND) as an in vivo carrier of siRNA to tumor tissues. We previously reported that a MEND containing YSK05 (YSK-MEND) efficiently delivered siRNA in RCC-bearing mice. We herein report on a combination therapy involving the use of siRNA-mediated specific gene knockdown and cytotoxic drug doxorubicin (DOX), and an advantage of YSK-MEND as an investigation tool for in vivo function of a gene. si-PLK1 encapsulated within YSK-MEND was prepared using the tertiary butanol dilution method. The in vitro cellular viability under the exposure of DOX was compared between OS-RC-2 cells with and without si-PLK1 transfection. In an in vivo study, tumor-bearing mice were systemically injected with YSK-MEND and DOX-loaded liposomes. The combination of DOX and si-PLK1 drastically reduced tumor growth rate, and apoptotic cells were observed. In an in vitro study, PLK1 knockdown increased G2/M cell population and reduced the expression of cyclin B1 (CCNB1) mRNA. CCNB1 suppression by si-PLK1 encapsulated in YSK-MEND was also observed in the in vivo experiments. A combination of DOX and anti-polo-like kinase 1 siRNA (si-PLK1) resulted in a measurable delay in OS-RC-2 tumor growth. This result suggests that the combination of si-PLK1 delivery and doxorubicin by YSK-MEND holds potential for RCC therapy via cell CCNB1 regulation.

An aptamer ligand based liposomal nanocarrier system that targets tumor endothelial cells

The objective of this study was to construct our recently developed aptamer-modified targeted liposome nano-carrier (Apt-PEG-LPs) system to target primary cultured mouse tumor endothelial cells (mTEC), both in vitro and in vivo. We first synthesized an aptamer-polyethylene glycol 2000-distearoyl phosphoethanolamine (Apt-PEG2000-DSPE). The conjugation of the Apt-PEG2000-DSPE was confirmed by MALDI-TOF mass spectroscopy. A lipid hydration method was used to prepare Apt-PEG-LPs, in which the outer surface of the PEG-spacer was decorated with the aptamer. Apt-PEG-LPs were significantly taken up by mTECs. Cellular uptake capacity was observed both quantitatively and qualitatively using spectrofluorometry, and confocal laser scanning microscopy (CLSM), respectively. In examining the extent of localization of aptamer-modified liposomes that entered the cells, approximately 39% of the Apt-PEG-LPs were not co-localized with lysotracker, indicating that they had escaped from endosomes. The uptake route involved a receptor mediated pathway, followed by clathrin mediated endocytosis. This Apt-PEG-LP was also applied for in vivo research whether this system could target tumor endothelial cells. Apt-PEG-LP and PEG5000-DSPE modified Apt-PEG-LP (Apt/PEG5000-LP) were investigated by human renal cell carcinoma (OS-RC-2 cells) inoculating mice using CLSM. Apt-PEG-LP and Apt/PEG5000-LP showed higher accumulation on tumor vasculature compared to PEG-LP and the co-localization efficacy of Apt-PEG-LP and Apt/PEG5000-LP on TEC were quantified 16% and 25% respectively, which was also better than PEG-LP (3%). The findings suggest that this system is considerable promise for targeting tumor endothelial cells to deliver drugs or genes in vitro and in vivo.

Advances in an active and passive targeting to tumor and adipose tissues

Introduction: Data reported during the last decade of the twentieth century indicate that passive targeting is an efficient strategy for delivering nanocarrier systems to tumor tissues. The focus of this review is on active targeting as a next-generation strategy for extending the capacity of a drug delivery system (DDS). Areas covered: Tumor vasculature targeting was achieved using arginine- glycine-aspartic acid, asparagine-glycine-arginine and other peptides, which are well-known peptides, as ligand against tumor vasculature. An efficient system for delivering small interfering RNA to the tumor vasculature involved the use of a multifunctional envelope-type nanodevice based on a pH-modified cationic lipid and targeting ligands. The active-targeting system was extended from tumor delivery to adipose tissue delivery, where endothelial cells are tightly linked and are impermeable to nanocarriers. In mice, prohibitin-targeted nanoparticles can be used to successfully deliver macromolecules to induce anti-obese effects. Finally, the successful delivery of nanocarriers to adipose tissue in obese mice via the enhanced permeability and retention-effect is reported, which can be achieved in tumor tissue. Expert opinion: Unlike tumor tissues, only a few reports have appeared on how liposomal carriers accumulate in adipose tissues after systemic injection. This finding, as well as active targeting to the adipose vasculature, promises to extend the capacity of DDS to adipose tissue. Since the site of action of nucleic acids is the cytosol, the intracellular trafficking of carriers and their cargoes as well as cellular uptake must be taken into consideration.

Remodeling of the Extracellular Matrix by Endothelial Cell-Targeting siRNA Improves the EPR-Based Delivery of 100 nm Particles.

A number of nano drug delivery systems have recently been developed for cancer treatment, most of which are based on the enhanced permeability and retention effect. The advantages of the enhanced permeability and retention effect can be attributed to immature vasculature. Herein we evaluated the intratumoral distribution of lipid nanoparticles when the VEGF receptor 2 on tumor endothelial cells was inhibited by liposomal siRNA. VEGF receptor 2 inhibition resulted in an increase in intratumoral distribution and therapeutic efficacy despite the maturation of the tumor vasculature. A small molecule inhibitor against matrix metalloproteinase and macrophage depletion cancelled the improvement in the distribution of the lipid nanoparticles, suggesting that remodeling of tumor microenvironment played a role in the facilitated intratumoral distribution via the down-regulation of VEGF receptor 2. Accordingly, our results suggest that the enhanced permeability and retention effect is dependent, not only on the structure of the tumor vasculature, but also on the dynamics of the tumor microenvironment including extracellular matrix remodeling. Regulating the tumor microenvironment and the extracellular matrix by delivering tumor endothelial cell-targeting siRNA could potentiate the enhanced permeability and retention effect-based strategy.A number of nano drug delivery systems have recently been developed for cancer treatment, most of which are based on the enhanced permeability and retention effect. The advantages of the enhanced permeability and retention effect can be attributed to immature vasculature. Herein we evaluated the intratumoral distribution of lipid nanoparticles when the VEGF receptor 2 on tumor endothelial cells was inhibited by liposomal siRNA. VEGF receptor 2 inhibition resulted in an increase in intratumoral distribution and therapeutic efficacy despite the maturation of the tumor vasculature. A small molecule inhibitor against matrix metalloproteinase and macrophage depletion cancelled the improvement in the distribution of the lipid nanoparticles, suggesting that remodeling of tumor microenvironment played a role in the facilitated intratumoral distribution via the down-regulation of VEGF receptor 2. Accordingly, our results suggest that the enhanced permeability and retention effect is dependent, not only on the structure of the tumor vasculature, but also on the dynamics of the tumor microenvironment including extracellular matrix remodeling. Regulating the tumor microenvironment and the extracellular matrix by delivering tumor endothelial cell-targeting siRNA could potentiate the enhanced permeability and retention effect-based strategy.

Effect of particle size on their accumulation in an inflammatory lesion in a dextran sulfate sodium (DSS)-induced colitis model

Taking advantage of the enhanced permeation and retention (EPR) effect is a promising approach for delivering macromolecules or nanoparticles to tumors. Recent studies revealed that this strategy is also applicable for targeting other pathological lesions (i.e. inflammatory disease). In the present study, we report the optimal size of a nanoparticle for allowing the higher accumulation of a particle in an inflammatory lesion using a dextran sulfate sodium (DSS)-induced colitis model. As a nanoparticle platform, we utilized a SS-cleavable and pH-activated lipid-like material (ssPalm), that can be used to produce particles in a variety of sizes ranging from 50nm to 180nm while using the same lipid composition. In healthy mice, particle accumulation remained low regardless of size. In contrast, the accumulation in inflammatory colon tissue was enhanced depending on the progress of the inflammation. In this situation, the apparent uptake clearance accumulation of a mid-sized particle (113nm on average) was higher than that for smaller and larger (54nm and 183nm in average, respectively) ones. Therefore, controlling particle size is an important parameter for the extensive targeting of inflammatory lesion.

Ligand density at the surface of a nanoparticle and different uptake mechanism: two important factors for successful siRNA delivery to liver endothelial cells.

The specific delivery of a gene to liver sinusoidal endothelial cells (LSEC) could become a useful strategy for treating various liver diseases associated with such cells. We previously reported that the accumulation of KLGR peptide modified liposomes through liver sinusoidal blood vessels was enhanced after an intravenous administration. Here, we report on an attempt to develop an LSEC targeted nanocarrier system to deliver siRNA for the successful knockdown of LSEC specific gene expression. The system involved the development of a multifunctional envelop-type nano device (MEND) modified with the KLGR peptide for siRNA delivery targeting LSEC. Our developed carrier successfully lowered specific gene expression in LSEC. An in vivo study showed that at a lower density of ligand at the surface of the MEND resulted in the highest knockdown of gene expression in LSEC. This is the first report of the successful delivery of siRNA to LSECs. Further experiments suggest that not only a higher endosomal escape efficiency into the cytosol but also the uptake mechanism as a function of ligand density are two important factors to be considered for targeting LSEC.