Hiroto Hatakeyama

Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid.

For successful cancer gene therapy via intravenous (i.v.) administration, it is essential to optimize the stability of carriers in the systemic circulation and the cellular association after the accumulation of the carrier in tumor tissue. However, a dilemma exists regarding the use of poly(ethylene glycol) (PEG), which is useful for conferring stability in the systemic circulation, but is undesirable for the cellular uptake and the following processes. We report the development of a PEG-peptide-lipid ternary conjugate (PEG-Peptide-DOPE conjugate (PPD)). In this strategy, the PEG is removed from the carriers via cleavage by a matrix metalloproteinase (MMP), which is specifically expressed in tumor tissues. An in vitro study revealed that the PPD-modified gene carrier (Multifunctional Envelope-type Nano Device: MEND) exhibited pDNA expression activity that was dependent on the MMP expression level in the host cells. In vivo studies further revealed that the PPD was potent in stabilizing MEND in the systemic circulation and facilitating tumor accumulation. Moreover, the i.v. administration of PPD or PEG/PPD dually-modified MEND resulted in the stimulation of pDNA expression in tumor tissue, as compared with a conventional PEG-modified MEND. Thus, MEND modified with PPD is a promising device, which has the potential to make in vivo cancer gene therapy achievable.

A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo

Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of both pDNA and siRNA. Modification of a MEND with polyuethylene glycol, i.e., PEGylation, is a potential strategy for in vivo delivery of MENDs to tumor tissue. However, PEGylation also inhibits both uptake and endosomal escape of MENDs. To overcome these limitations, we developed a PEG-peptide-DOPE (PPD) that can be cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, to further improve the silencing activity of encapsulated siRNA, we modified the PPD-MEND with a pH-sensitive fusogenic GALA peptide (GALA/PPD-MEND). First, we determined the GALA and PPD content that would optimize the synergistic functions of GALA and PPD. The most efficient gene silencing activity was achieved when GALA and either conventional PEG-lipid or PPD were used to modify the MEND at a molar ratio of 1:1. In this case, the silencing activity was comparable to that achieved when using a MEND that had not been modified with PEG (unmodified MEND). Furthermore, in vivo topical administration revealed that optimized PPD/GALA-MENDa resulted in more efficient gene silencing compared with unmodified MENDs. Collectively, data demonstrate that introduction of both of a pH-sensitive fusogenic GALA peptide and PPD into the MEND facilitates nanoparticle endosomal escape, thereby enhancing the efficiency of siRNA delivery and gene silencing.

Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid

Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of nucleic acids. For tumor delivery of a MEND, PEGylation is a useful method, which confers a longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. To overcome this, we developed a PEG-peptide-DOPE (PPD) that is cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, we report on the systemic delivery of siRNA to tumors by employing a MEND that is modified with PPD (PPD-MEND). An in vitro study revealed that PPD modification accelerated both cellular uptake and endosomal escape, compared to a conventional PEG modified MEND. To balance both systemic stability and efficient activity, PPD-MEND was further co-modified with PEG-DSPE. As a result, the systemic administration of the optimized PPD-MEND resulted in an approximately 70% silencing activity in tumors, compared to non-treatment. Finally, a safety evaluation showed that the PPD-MEND showed no hepatotoxicity and innate immune stimulation. Furthermore, in a DNA microarray analysis in liver and spleen tissue, less gene alternation was found for the PPD-MEND compared to that for the PEG-unmodified MEND due to less accumulation in liver and spleen.

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.

Dual-ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery

The objective of this study was to develop an efficient dual-ligand based PEGylated liposomal delivery system that had target specificity as well as properties that would enhance cellular uptake. PEGylated liposomes (PEG-LP) were prepared by the lipid film hydration method by adding distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG2000) to a lipid mixture. The cyclic RGD (Arg-Gly-Asp) peptide, a specific ligand with affinity for Integrin α(v)β(3) was coupled to the distal end of the PEG on the PEG-LP (RGD-PEG-LP). Stearylated octaarginine (STR-R8) was incorporated on the surface of the RGD-PEG-LP as dual-ligand (R8/RGD-PEG-LP) that functions as a cell penetrating peptide (CPP). RGD-PEG-LP and R8/RGD-PEG-LP were preferentially taken up by caveolae-mediated and clathrin-mediated endocytosis pathways, respectively. Compared to PEG-LP, R8/RGD-PEG-LP showed an enhanced cellular uptake as well as a higher transfection efficiency in Integrin α(v)β(3) expressing cells. However, the amount of cellular uptake or gene expression by the single ligand versions was negligible, even in Integrin α(v)β(3) expressing cells. No remarkable difference in cellular uptake or gene expression was observed for cells in which the expression of targeted receptors was absent. It can be concluded that dual-ligand modified PEG-LP possesses a strong capability for the efficient internalization of PEG-LP and consequently would be an effective tool for the targeted delivery of macromolecules or chemotherapeutics through accelerated cellular uptake.

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.

Size-controlled, dual-ligand modified liposomes that target the tumor vasculature show promise for use in drug-resistant cancer therapy.

Anti-angiogenic therapy is a potential chemotherapeutic strategy for the treatment of drug resistant cancers. However, a method for delivering such drugs to tumor endothelial cells remains to be a major impediment to the success of anti-angiogenesis therapy. We designed liposomes (LPs) with controlled diameter of around 300 nm, and modified them with a specific ligand and a cell penetrating peptide (CPP) (a dual-ligand LP) for targeting CD13-expressing neovasculature in a renal cell carcinoma (RCC). We modified the LPs with an NGR motif peptide on the top of poly(ethylene glycol) and tetra-arginine (R4) on the surface of the liposome membrane as a specific and CPP ligand, respectively. The large size prevented extravasation of the dual-ligand LP, which allowed it to associate with target vasculature. While a single modification with either the specific or CPP ligand showed no increase in targetability, the dual-ligand enhanced the amount of delivered liposomes after systemic administration to OS-RC-2 xenograft mice. The anti-tumor activity of a dual-ligand LP encapsulating doxorubicin was evaluated and the results were compared with Doxil, which is clinically used to target tumor cells. Even though Doxil showed no anti-tumor activity, the dual-ligand LP suppressed tumor growth because the disruption of tumor vessels was efficiently induced. The comparison showed that tumor endothelial cells (TECs) were more sensitive to doxorubicin by 2 orders than RCC tumor cells, and the disruption of tumor vessels was efficiently induced. Collectively, the dual-ligand LP is promising carrier for the treatment of drug resistant RCC via the disruption of TECs.

Cancer multidrug resistance: mechanisms involved and strategies for circumvention using a drug delivery system.

Multidrug resistance (MDR), the principal mechanism by which many cancers develop resistance to chemotherapy, is one of the major obstacles to the successful clinical treatment of various types of cancer. Several key regulators are responsible for mediating MDR, a process that renders chemotherapeutic drugs ineffective in the internal organelles of target cells. A nanoparticulate drug delivery system (DDS) is a potentially promising tool for circumventing such MDR, which can be achieved by targeting tumor cells themselves or tumor endothelial cells that support the survival of MDR cancer cells. The present article discusses key factors that are responsible for MDR in cancer cells, with a specific focus on the application of DDS to overcome MDR via the use of chemotherapy or macromolecules.

The effect of liposomal size on the targeted delivery of doxorubicin to Integrin αvβ3-expressing tumor endothelial cells

Size of the liposomes (LPs) specially governs its biodistribution. In this study, LPs were developed with controlled sizes, where variation in LP size dictates the ligand-receptor interaction, cellular internalization and its distribution within the tumor microenvironment. The therapeutic efficacies of doxorubicin (DOX)-loaded RGD modified small size (~100 nm in diameter, dnm) and large size (~300 dnm) PEGylated LPs (RGD-PEG-LPs) were compared to that of Doxil (a clinically used DOX-loaded PEG-LP, ~100 dnm) in DOX resistant OSRC-2 (Renal cell carcinoma, RCC) tumor xenografts. Doxil, which accumulated in tumor tissue via the enhanced permeability and retention (EPR) effect, failed to suppress tumor growth. Small size RGD-PEG-LP, that targets the tumor endothelial cells (TECs) and extravasates to tumor cells, failed to provide anti-tumor effect. Large size RGD-PEG-LP preferentially targets the TECs via minimization of the EPR effect, and significantly reduced the tumor growth, which was exerted through its strong anti-angiogenic activity on the tumor vasculature rather than having a direct effect on DOX resistant RCC. The prepared large size RGD-PEG-LP that targets the TECs via interacting with Integrin αvβ3, is a potentially effective and alternate therapeutic strategy for the treatment of DOX resistant tumor cells by utilizing DOX, in cases where Doxil is ineffective.

Design of a dual-ligand system using a specific ligand and cell penetrating peptide, resulting in a synergistic effect on selectivity and cellular uptake

In this study, a dual-ligand liposomal system comprised of a specific ligand and a cell penetrating peptide (CPP) is described to enhance selectivity and cellular uptake. Dual-ligand PEGylated liposomes were prepared by modifying the end of the PEG with an NGR motif peptide, followed by a surface coating of the liposomes with stearylated oligoarginine (STR-RX). The NGR motif recognizes CD13, a marker protein located on tumor endothelial cells. A suitable number of RX units was determined to be R4, since it can be masked by the PEG aqueous layer. Although no enhanced cellular uptake was observed when a single modification of PEGylated liposomes with either NGR- or STR-R4 was used, the dual-modification with NGR and STR-R4 stimulated uptake of PEGylated liposomes by CD13 positive cells, and this uptake was superior to that obtained by PEG-unmodified liposomes modified with STR-R4. The dual-ligand system shows a synergistic effect on cellular uptake. Collectively, the dual-ligand system promises to be useful in the development efficient and specific drug delivery systems.