Effective Therapy Using a Liposomal siRNA that Targets the Tumor Vasculature in a Model Murine Breast Cancer with Lung Metastasis.

Although metastatic cancer is a major cause of death for cancer patients, no efficacious treatment for metastasis is available. We previously showed that the growth of a primary tumor could be inhibited by the administration of an anti-angiogenic small interfering RNA (siRNA) that is encapsulated in an RGD peptide-modified lipid nanoparticle (RGD-LNP). We herein report on the delivery of siRNA by an RGD-LNP to the vasculature is also effective for treating metastatic tumors. We compared the RGD-LNP with the polyethylene glycol (PEG)ylated LNP (PEG-LNP) in terms of accumulation in a lung-metastasized model. Despite malformed structure of vasculature in the metastasized lung, the accumulation of the PEG-LNP in the metastasized lung was lower than that for the RGD-LNP, which suggests that the delivery strategy based on vascular permeability is not completely effective for targeting metastasis tumors. The systemic injection of the RGD-LNP induced a significant silencing in the metastasized vasculature, but not in the normal lung. In addition, the continuous injection of the RGD-LNP encapsulating siRNA against a delta-like ligand 4 (DLL4) drastically prolonged the overall survival of metastasized model mice. Accordingly, our current findings suggest that vasculature targeting would be more effective than enhanced permeability and retention effect-based therapy for the treatment of metastatic cancer.

Efficient siRNA Delivery by Lipid Nanoparticles Modified with a Nonstandard Macrocyclic Peptide for EpCAM-Targeting.

The development of a specific, effective method for the delivery of therapeutics including small molecules and nucleic acids to tumor tissue remains to be solved. Numerous types of lipid nanoparticles (LNPs) have been developed in attempts to achieve this goal. However, LNPs are probably not taken up by target cells because cancer-targeting LNPs are typically modified with poly(ethylene glycol) (PEG), which inhibits the cellular uptake of LNPs, to passively accumulate in tumor tissue via the enhanced permeability and retention (EPR) effect. It would clearly be important to develop a LNP with both a prolonged circulation and cancer-specific efficient uptake for use in an innovative nanodrug delivery system. Herein, we assessed the effect of nonstandard macrocyclic peptides against the epithelial cell adhesion molecule (EpCAM) Epi-1, which was discovered by a random nonstandard peptides integrated discovery (RaPID) system, on the cellular uptake and therapeutics delivery of LNPs. A liposomal siRNA delivery system (MEND) was modified with an Epi-1 lipid-derivative (EpCAM-targeting MEND; ET-MEND). The resulting ET-MEND showed a more than 27-fold increase in cellular uptake in EpCAM-positive cell lines. In the case of negative cells, cellular uptake and the efficiency of the ET-MEND for delivering therapeutics were comparable with those of nonmodified MEND. In addition, when systemically injected, the ET-MEND successfully inhibited gene expression in the tumor tissue at a dose of 0.5 mg siRNA/kg without any obvious toxicity. These results suggest that a combination of a specific peptide ligand can be used to identify a RaPID system and that the use of such a MEND for liposomal drug delivery has the potential for use in developing a system for the efficacious delivery of pharmaceuticals to various cancer cells.

Modality of tumor endothelial VEGFR2 silencing-mediated improvement in intratumoral distribution of lipid nanoparticles.

The vascular endothelial growth factor (VEGF)-mediated enhancement in vascular permeability is considered to be a major factor in tumor-targeting delivery via the enhanced permeability and retention (EPR) effect. We previously reported that the silencing of the endothelial VEGF receptor (VEGFR2) by a liposomal siRNA system (RGD-MEND) resulted in an enhanced intratumoral distribution of polyethylene glycol (PEG)-modified liposomes (LPs) in a renal cell carcinoma, a type of hypervascularized cancer, although the inhibition of VEGF signaling would be expected to decrease the permeability of the tumor vasculature. We herein report that the enhancement in the intratumoral distribution of LPs by VEGFR2 inhibition was dependent on the vascular type of the tumor (stroma vessel type; SV and tumor vessel type; TV). In the case of TV-type tumors (renal cell carcinoma and hepatocellular carcinoma), inhibiting VEGFR2 improved intratumoral distribution, while no effect was found in the case of SV-type tumors (colorectal cancer). Moreover, through a comparison of the intratumoral distribution of LPs with a variety of physical properties (100nm vs 400nm, neutral vs negative vs positive), VEGFR2 inhibition was found to alter the tumor microenvironment, including heparan sulfate proteoglycans (HSPGs). In addition, the results regarding the effect of the size of nanoparticles indicated that VEGFR2 inhibition improved the penetration of nanoparticles through the vessel wall, but not via permeability, suggesting the involvement of an unknown mechanism. Our findings suggest that a combination of anti-angiogenic therapy and delivery via the EPR effect would be useful in certain cases, and that altering the tumor microenvironment by VEGFR2 blockade has a drastic effect on the intratumoral distribution of nanoparticles.

Scalable preparation of poly(ethylene glycol)-grafted siRNA-loaded lipid nanoparticles using a commercially available fluidic device and tangential flow filtration.

While a number of siRNA delivery systems have been developed, the methods used in their preparation are not suitable for large-scale production. We herein report on methodology for the large-scale preparation of liposomal siRNA using a fluidic device and tangential flow filtration (TFF). A number of studies have appeared on the use of fluidic devices for preparing and purifying liposomes, but no systematic information regarding appropriate membrane type of commercially available apparatus is available. The findings reported herein indicate that, under optimized conditions, a microfluidic device and TFF can be used to produce siRNA lipid nanoparticles with the same characteristics as traditional ones'. The in vivo silencing efficiency of these lipid nanoparticles in the liver was comparable to laboratory-produced nanoparticles. In addition, con-focal laser scanning microscopy analyses revealed that they accumulated in the liver accumulation at the same levels as particles produced by batch-type and continuous-type procedures. This methodology has the potential to contribute to the advancement of this process from basic research to clinical studies of liposomal DDS.

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.

Preparation of a Cyclic RGD: Modified Liposomal SiRNA Formulation for Use in Active Targeting to Tumor and Tumor Endothelial Cells.

The delivery of SiRNA is not only a challenging strategy for developing new remedies, but is also useful as an analytic tool for an in vivo phenotypic alteration by loss-of-function. Specifically, ligand-mediated SiRNA active targeting can be used to silence any gene in any organ of interest. In this chapter, we describe the preparation of an active targeting system to tumor endothelial cells (TECs) using liposomal SiRNA modified with cyclic RGD peptides. The procedure consists of essentially three steps: (1) the synthesis of a cyclic RGD peptide derivative, (2) SiRNA encapsulation into a liposomal delivery system, and (3) modification of liposomal SiRNA with a cyclic RGD derivative.

Optimization of a siRNA Carrier Modified with a pH-Sensitive Cationic Lipid and a Cyclic RGD Peptide for Efficiently Targeting Tumor Endothelial Cells.

In recent years, anti-angiogenic therapy has attracted much interest because it is a versatile approach to treating most types of tumors, and therefore would be expected to be applicable for various cancers. Severe adverse events in patients treated with currently available anti-angiogenic therapeutics have, however, been reported, and these are caused by their inhibitory effects in normal tissue. To achieve an efficient anti-angiogenic therapy with minimal toxicity, a drug delivery system (DDS) specific to tumor endothelial cells (TECs) is needed. Cyclic RGD (cRGD) is a well-known ligand against αVß3 integrin that is expressed at high levels in the cell surface of TECs. To address this issue, we previously developed a cyclic RGD-equipped liposomal DDS (RGD-MEND) in which small interfering RNA (siRNA) was encapsulated. However, in the previous study, details of the preparation steps were not thoroughly examined. In this paper, to produce the most efficient delivery of therapeutic TECs, we explored optimum preparation conditions and components of the RGD-MEND. The cellular uptake and silencing ability of the RGD-MEND were investigated as a function of ligand density, poly(ethyleneglycol) linker length, and lipid composition. As a result, a knockdown efficiency that was five-fold higher than that of the previously reported one (ED50, from 4.0 to 0.75 mg/kg) was achieved.

Efficient Packaging of Plasmid DNA Using a pH Sensitive Cationic Lipid for Delivery to Hepatocytes.

Plasmid DNA (pDNA) is expected to be a new class of medicine for treating currently incurable diseases. To deliver these nucleic acids, we developed a liposomal delivery system we have called a multifunctional envelope-type nano device (MEND). In this report, we demonstrate that a MEND containing a pH-sensitive cationic lipid, YSK05 (YSK-MEND), efficiently delivered pDNA via systemic injection, and that its expression was highly dependent on the encapsulation state of the pDNA. In the preparation, the pH, ionic strength, and sodium chloride (NaCl) concentration of the lipid/pDNA mixture strongly affected the encapsulation efficiency of pDNA. Additionally, the transgene expression of luciferase in the liver by the injected YSK-MEND was dependent on the encapsulation state of pDNA rather than the nature of the YSK-MEND. Confocal laser scanning microscopy findings revealed that injection of the YSK-MEND led to homogenous gene expression in the liver compared to injection via the hydrodynamic tail vein (HTV). Concerning the safety of the YSK-MEND, a transient increase in the activity of liver enzymes was observed. However, no significant adverse events were observed. Taken together, the YSK-MEND represents a potentially attractive therapy for the treatment of various hepatic diseases.