Eiji Yuba

A liposome-based antigen delivery system using pH-sensitive fusogenic polymers for cancer immunotherapy.

Highly pH-sensitive liposomes that deliver antigenic molecules into cytosol through fusion with or destabilization of endosome were prepared by surface modification of egg yolk phosphatidylcholine/dioleoylphosphatidylethanolamine (1/1, mol/mol) liposomes with 3-methylglutarylated poly(glycidol) of linear (MGlu-LPG) or hyperbranched structure (MGlu-HPG). These polymer-modified liposomes were stable at neutral pH, but they became strongly destabilized below pH 6, which corresponds to the pH of endosome. These polymer-modified liposomes were taken up by murine dendritic cells (DCs) more efficiently than the unmodified liposomes were through an endocytic pathway. They introduced entrapped ovalbumin (OVA) molecules into cytosol. Subcutaneous or nasal administration of the polymer-modified liposomes loaded with OVA induced generation of OVA-specific cytotoxic T cells (CTL) much more effectively than the unmodified liposomes loaded with OVA. Furthermore, administration of the polymer-modified OVA-loaded liposomes to mice bearing E.G7-OVA tumor significantly reduced the tumor burden, although the OVA-loaded unmodified liposomes only slightly affected tumor growth. Results suggest that the polymer-modified liposomes with highly pH-sensitive destabilizing property are promising as antigen carriers for efficient cancer immunotherapy.

PEGylated PAMAM dendrimer-doxorubicin conjugate-hybridized gold nanorod for combined photothermal-chemotherapy.

We prepared pH-sensitive drug-dendrimer conjugate-hybridized gold nanorod as a promising platform for combined cancer photothermal-chemotherapy under in vitro and in vivo conditions. Poly(ethylene glycol)-attached PAMAM G4 dendrimers (PEG-PAMAM) were first covalently linked on the surface of mercaptohexadecanoic acid-functionalized gold nanorod (MHA-AuNR), with subsequent conjugation of anti-cancer drug doxorubicin (DOX) to dendrimer layer using an acid-labile-hydrazone linkage to afford PEG-DOX-PAMAM-AuNR particles. The particles with a high PEG-PAMAM dendrimer coverage density (0.28 per nm(2) AuNR) showed uniform sizes and excellent colloidal stability. In vitro drug release studies demonstrated that DOX released from PEG-DOX-PAMAM-AuNR was negligible under normal physiological pH, but it was enhanced significantly at a weak acidic pH value. The efficient intracellular acid-triggered DOX release inside of lysosomes was confirmed using confocal laser scanning microscopy analysis. Furthermore, the combined photothermal-chemo treatment of cancer cells using PEG-DOX-PAMAM-AuNR for synergistic hyperthermia ablation and chemotherapy was demonstrated both in vitro and in vivo to exhibit higher therapeutic efficacy than either single treatment alone, underscoring the great potential of PEG-DOX-PAMAM-AuNR particles for cancer therapy.

Multi-functional liposomes having temperature-triggered release and magnetic resonance imaging for tumor-specific chemotherapy.

For development of tumor-specific chemotherapy, we designed liposomes with temperature-triggered drug release and magnetic resonance imaging (MRI) functions. We prepared multi-functional liposomes by incorporating thermosensitive poly(2-ethoxy(ethoxyethyl)vinyl ether) chains with a lower critical solution temperatures around 40 °C and polyamidoamine G3 dendron-based lipids having Gd(3+) chelate residues into pegylated liposomes. These stable doxorubicin (DOX)-loaded liposomes retained DOX in their interior below physiological temperature but released DOX immediately at temperatures greater than 40 °C. They exhibited excellent ability to shorten the longitudinal proton relaxation time. When administered intravenously into colon 26 tumor-bearing mice, accumulated liposomes in tumors increased with time, reaching a constant level 8 h after administration by following T(1)-weighted MRI signal intensity in tumors. Liposome size affected the liposome accumulation efficiency in tumors: liposomes of about 100 nm diameter were accumulated more efficiently than those with about 50 nm diameter. Tumor size also affected accumulation: more efficient accumulation occurred in larger tumors. Tumor growth was strongly suppressed when liposomes loaded with DOX were administered intravenously into tumor-bearing mice and the tumor was heated mildly at 44 °C for 10 min at 8 h after administration. Multi-functional liposomes having temperature-triggered drug release and MRI functions might engender personalized chemotherapy, providing efficient patient-optimized chemotherapy.

pH-Sensitive fusogenic polymer-modified liposomes as a carrier of antigenic proteins for activation of cellular immunity

By modification of liposomes with poly(glycidol) derivatives such as succinylated poly(glycidol) and 3-methylglutarylated poly(glycidol), we have developed functional liposomes that generate fusion ability at mildly acidic pH. We investigated the feasibility of these polymer-modified liposomes as a carrier of antigenic proteins for induction of cellular immunity. These pH-sensitive fusogenic liposomes encapsulating ovalbumin (OVA) were applied to DC2.4 cells, a murine dendritic cell line. Observation with confocal laser scanning microscopy showed that these polymer-modified liposomes were taken up efficiently by the cells, thereafter delivering their contents into the cytosol, probably through fusion with endosomal membranes. Murine bone marrow-derived dendritic cells treated with polymer-modified liposomes encapsulating OVA stimulated CD8-OVA1.3 cells more strongly than OT4H.1D5 cells, indicating that the liposomes induced MHC class I-restricted presentation. Furthermore, administration of the polymer-modified, OVA-loaded liposomes from nasal cavities of mice induced stronger cellular immune responses than the OVA-loaded plain liposomes. Because the ability of the polymer-modified liposomes to activate cellular immunity was comparable to that of Freunds complete adjuvant, which is a widely used adjuvant, they potentially have use in production of efficient vaccines for immunotherapy.

Carboxylated hyperbranched poly(glycidol)s for preparation of pH-sensitive liposomes

Previous reports by the authors described intracellular delivery using liposomes modified with various carboxylated poly(glycidol) derivatives. These linear polymer-modified liposomes exhibited a pH-dependent membrane fusion behavior in cellular acidic compartments. However, the effect of the backbone structure on membrane fusion activity remains unknown. Therefore, this study specifically investigated the backbone structure to obtain pH-sensitive polymers with much higher fusogenic activity and to reveal the effect of the polymer backbone structure on the interaction with the membrane. Hyperbranched poly(glycidol) (HPG) derivatives were prepared as a new type of pH-sensitive polymer and used for the modification of liposomes. The resultant HPG derivatives exhibited high hydrophobicity and intensive interaction with the membrane concomitantly with the increasing degree of polymerization (DP). Furthermore, HPG derivatives showed a stronger interaction with the membrane than the linear polymers show. Liposomes modified with HPG derivatives of high DP delivered contents into the cytosol of DC2.4 cells, a dendritic cell line, more effectively than the linear polymer-modified liposomes do. Results show that the backbone structure of pH-sensitive polymers affected their pH-sensitivity and interaction with liposomal and cellular membranes.

Potentiation of pH-sensitive polymer-modified liposomes with cationic lipid inclusion as antigen delivery carriers for cancer immunotherapy.

Cationic lipid-incorporated liposomes modified with pH-sensitive polymers were prepared by introducing 3, 5-didodecyloxybenzamidine as a cationic lipid to egg yolk phosphatidylcholine liposomes modified with 3-methylglutarylated hyperbranched poly(glycidol) (MGlu-HPG) as a pH-sensitive polymer. These liposomes were stable at neutral pH, but were destabilized below pH 6.0 because MGlu-HPG changed its characteristics from hydrophilic to hydrophobic in response to the pH decrease. Cationic lipid inclusion improved their pH sensitivity at weakly acidic pH and association of liposomes with murine dendritic cell (DC) lines. Cationic lipid-incorporated liposomes delivered entrapped ovalbumin (OVA) molecules not only to cytosol but also to endosome/lysosome. Treatment with cationic lipid-incorporated liposomes induced up-regulation of antigen presentation-involved molecules on DCs, the promotion of cytokine production, and antigen presentation via both major histocompatibility complex (MHC) class I and II molecules. Especially, antigen presentation via MHC class II was promoted by cationic lipid inclusion, which might correspond to efficient endosome/lysosome delivery of OVA. Subcutaneous administration of OVA-loaded cationic lipid-incorporated liposomes induced antigen-specific antibody production in serum and Th1-dominant immune responses in the spleen. Furthermore, administration of the cationic lipid-incorporated liposomes to mice bearing E.G7-OVA tumor more significantly reduced the tumor volume than liposomes without cationic lipids. Therefore, cationic lipid inclusion into pH-sensitive polymer-modified liposomes, which can achieve both efficient antigen intracellular delivery and activation of antigen presenting cell, is an effective approach to develop antigen carriers for efficient cancer immunotherapy.

Dextran derivative-based pH-sensitive liposomes for cancer immunotherapy.

pH-Sensitive dextran derivatives having 3-methylglutarylated residues (MGlu-Dex) were prepared by reacting dextran with 3-methyl-glutaric anhydride. MGlu-Dex changed the protonation state and their characteristics from hydrophilic to hydrophobic in neutral and acidic pH regions. Surface modification of egg yolk phosphatidylcholine liposomes with MGlu-Dex produced highly pH-sensitive liposomes that were stable at neutral pH but which were destabilized strongly in the weakly acidic pH region. MGlu-Dex-modified liposomes were taken up efficiently by dendritic cells and delivered entrapped ovalbumin (OVA) molecules into the cytosol. When MGlu-Dex-modified liposomes loaded with OVA were administered subcutaneously to mice, the antigen-specific humoral and cellular immunity was induced more effectively than the unmodified liposomes loaded with OVA. Furthermore, administration of MGlu-Dex-modified liposomes loaded with OVA to mice bearing E.G7-OVA tumor significantly suppressed tumor growth and extended the mice survival. Results suggest that MGlu-Dex-modified liposomes are promising for the production of safe and potent antigen delivery systems that contribute to the establishment of efficient cancer immunotherapy.

Gene delivery to dendritic cells mediated by complexes of lipoplexes and pH-sensitive fusogenic polymer-modified liposomes.

Dendritic cells (DCs) are potent professional antigen presenting cells that are useful for cancer immunotherapy. We previously reported the preparation and characterization of complexes of lipoplexes with pH-sensitive fusogenic liposomes, which comprise polymers based on poly(glycidol) with carboxyl groups, to transfect various malignant cell lines. The present study applied this kind of vectors to transfection of a murine DC line DC2.4. We first optimized the ratios of their components for efficient transfection. We subsequently investigated the effects of ligands and pH-sensitive polymers to improve transfection activities. Our results indicate that the anionic surface derived from pH-sensitive polymers might be recognized by scavenger receptors on DC2.4 cells. In addition, no effects on transfection or cell association were observed by attaching ligands such as transferrin and mannan. We found that more sensitive pH-responding polymers led to higher transfection activities into DC2.4 cells, which suggest that endosomal escape is important for transfection into DC2.4 cells. These complexes with pH-sensitive fusogenic polymers exhibited higher transfection activity toward DC2.4 cells than some commercial reagents and hence may be useful as a gene vector for DCs.

Preparation and characterization of complexes of liposomes with gold nanoparticles

Gold nanoparticles (Au NPs), which are extremely useful materials for imaging and photothermal therapy, typically require a drug delivery system to transport them to the affected tissue and into the cells. Since liposomes are approved as drug carriers, complexes of liposomes with Au NPs were considered ideal solutions to deliver Au NPs to the target site in vivo. In this study, we prepared complexes of various liposomes with Au NPs via physical absorption and characterized them. The time dependency of the surface plasmon resonance of this complex, which is a unique property of Au NPs, shows that the liposomes promote the formation of stable dispersions of Au NPs under isotonic conditions, even though intact Au NPs aggregate immediately. From a release assay of calcein from liposomes and transmission electron microscopy analysis, the Au NPs were complexed with liposomes without membrane disruption. These complexes could be formed by using cationic liposomes and polyethylene glycol-modified liposomes, as well as by using phosphatidylcholine liposomes, which are useful for drug and gene delivery. We proposed this kind of complex as a nanomedicine with diagnostic and therapeutic ability.

Carbon nanotube–liposome supramolecular nanotrains for intelligent molecular-transport systems

Biological network systems, such as inter- and intra-cellular signalling systems, are handled in a sophisticated manner by the transport of molecular information. Over the past few decades, there has been a growing interest in the development of synthetic molecular-transport systems. However, several key technologies have not been sufficiently realized to achieve optimum performance of transportation methods. Here we show that a new type of supramolecular system comprising of carbon nanotubes and liposomes enables the directional transport and controlled release of carrier molecules, and allows an enzymatic reaction at a desired area. The study highlights important progress that has been made towards the development of biomimetic molecular-transport systems and various lab-on-a-chip applications, such as medical diagnosis, sensors, bionic computers and artificial biological networks.