Dietary Gamma-Aminobutyric Acid (GABA) Induces Satiation by Enhancing the Postprandial Activation of Vagal Afferent Nerves.

Gamma-aminobutyric acid (GABA) is present in the mammalian brain as the main inhibitory neurotransmitter and in foods. It is widely used as a supplement that regulates brain function through stress-reducing and sleep-enhancing effects. However, its underlying mechanisms remain poorly understood, as it is reportedly unable to cross the blood-brain barrier. Here, we explored whether a single peroral administration of GABA affects feeding behavior as an evaluation of brain function and the involvement of vagal afferent nerves. Peroral GABA at 20 and 200 mg/kg immediately before refeeding suppressed short-term food intake without aversive behaviors in mice. However, GABA administration 30 min before refeeding demonstrated no effects. A rise in circulating GABA concentrations by the peroral administration of 200 mg/kg GABA was similar to that by the intraperitoneal injection of 20 mg/kg GABA, which did not alter feeding. The feeding suppression by peroral GABA was blunted by the denervation of vagal afferents. Unexpectedly, peroral GABA alone did not alter vagal afferent activities histologically. The coadministration of a liquid diet and GABA potentiated the postprandial activation of vagal afferents, thereby enhancing postprandial satiation. In conclusion, dietary GABA activates vagal afferents in collaboration with meals or meal-evoked factors and regulates brain function including feeding behavior.

Chondroitin Sulfate-Based pH-Sensitive Polymer-Modified Liposomes for Intracellular Antigen Delivery and Induction of Cancer Immunity.

Induction of cancer-specific cytotoxic T lymphocytes is crucially important to complement therapeutic effects of immune checkpoint inhibitors and to achieve efficient cancer immunotherapy. To induce cancer-specific cytotoxic T lymphocytes, cancer antigen carriers must have multiple functions to deliver cancer antigens to antigen presenting cells, release antigens into cytosol, and promote the maturation of these cells. We earlier achieved cytosolic delivery of antigens and induction of antigen-specific cytotoxic T lymphocytes using carboxylated polyglycidol or polysaccharide derivative-modified liposomes that can induce membrane fusion with endosomes in response to weakly acidic pH. Furthermore, pH-sensitivity and adjuvant properties of these polymers were enhanced strongly by introduction of hydrophobic carboxylated units to dextran. Against our expectations, these polymer-modified liposomes only slightly induce cancer immunity, probably because of the high hydrophobicity of spacer units. This study used a polysaccharide with charged groups (chondroitin sulfate) instead of dextran as a backbone to reduce hydrophobicity. Chondroitin sulfate derivative-modified liposomes showed almost equal pH-sensitivity to that of dextran derivative-modified liposomes and achieved selective delivery to dendritic cells, whereas dextran derivative-modified liposomes were highly taken up by both dendritic cells and fibroblasts. Chondroitin sulfate derivative-modified liposomes delivered model antigenic proteins into cytosol of dendritic cells and promoted cytokine production from the cells, leading to tumor regression on tumor-bearing mice after subcutaneous administration. Results demonstrate that charged groups having polysaccharide as a backbone can be used in an effective strategy to balance strong hydrophobicity of spacer units with their utilization for immunity-inducing systems.

Development of pH-Responsive Hyaluronic Acid-Based Antigen Carriers for Induction of Antigen-Specific Cellular Immune Responses.

Cancer immunotherapy has gained much attention because of the recent success of immune checkpoint inhibitors. Nevertheless, clinical therapeutic effects of immune checkpoint inhibitors remain limited, probably because most patients have other immune checkpoint molecules or because they lack cancer-specific cytotoxic T lymphocytes. Induction of cancer-specific cytotoxic T lymphocytes requires efficient antigen delivery systems that can convey cancer antigens specifically to antigen presenting cells, promote the endosomal escape of antigen into cytosol, and activate immune cells. Earlier, we reported cytoplasmic delivery systems of antigen using pH-sensitive polymer-modified liposomes. Adjuvant molecules were further incorporated into these liposomes to provide activation properties of cellular immune responses. This study further introduced cell specificity to these liposomal systems using hyaluronic acid-based pH-sensitive polymers, which are recognized by CD44 expressing on antigen presenting cells. pH-Sensitive hyaluronic acid derivative-modified liposomes showed much higher cellular association to antigen presenting cells than to fibroblasts with less CD44 expression. These liposomes achieved the delivery of model antigenic proteins into cytosol of dendritic cells and promoted Th1 cytokine production from the cells. Subcutaneous administration of these liposomes to mice induced antigen-specific cellular immune response in the spleen, leading to tumor regression in tumor-bearing mice. The results show that pH-sensitive hyaluronic acid derivative-modified liposomes are promising as multifunctional antigen carriers having cell-specificity, cytoplasmic antigen delivery performance, and adjuvant property to induce antigen-specific cellular immunity.

Hyaluronic Acid-Based pH-Sensitive Polymer-Modified Liposomes for Cell-Specific Intracellular Drug Delivery Systems.

For the enhancement of therapeutic effects and reduction of side effects derived from anticancer drugs in cancer chemotherapy, it is imperative to develop drug delivery systems with cancer-specificity and controlled release function inside cancer cells. pH-sensitive liposomes are useful as an intracellular drug delivery system because of their abilities to transfer their contents into the cell interior through fusion or destabilization of endosome, which has weakly acidic environment. We earlier reported liposomes modified with various types of pH-sensitive polymers based on synthetic polymers and biopolymers as vehicles for intracellular drug delivery systems. In this study, hyaluronic acid (HA)-based pH-sensitive polymers were designed as multifunctional polymers having not only pH-sensitivity but also targeting properties to cells expressing CD44, which is known as a cancer cell surface marker. Carboxyl group-introduced HA derivatives of two types, MGlu-HA and CHex-HA, which have a more hydrophobic side chain structure than that of MGlu-HA, were synthesized by reaction with various dicarboxylic anhydrides. These polymer-modified liposomes were stable at neutral pH, but showed content release under weakly acidic conditions. CHex-HA-modified liposomes delivered their contents into CD44-expressing cells more efficiently than HA-modified or MGlu-HA-modified liposomes or unmodified liposomes, whereas the same liposomes were taken up only slightly by cells expressing CD44 proteins less. Competition assay using free HA or other polymers revealed that HA derivative-modified liposomes might be recognized by CD44. Therefore, HA-derivative-modified liposomes are useful as cell-specific intracellular drug delivery systems.

pH-Responsive Micelle-Based Cytoplasmic Delivery System for Induction of Cellular Immunity.

(1) Background: Cytoplasmic delivery of antigens is crucial for the induction of cellular immunity, which is an important immune response for the treatment of cancer and infectious diseases. To date, fusogenic protein-incorporated liposomes and pH-responsive polymer-modified liposomes have been used to achieve cytoplasmic delivery of antigen via membrane rupture or fusion with endosomes. However, a more versatile cytoplasmic delivery system is desired for practical use. For this study, we developed pH-responsive micelles composed of dilauroyl phosphatidylcholine (DLPC) and deoxycholic acid and investigated their cytoplasmic delivery performance and immunity-inducing capability. (2) Methods: Interaction of micelles with fluorescence dye-loaded liposomes, intracellular distribution of micelles, and antigenic proteins were observed. Finally, antigen-specific cellular immune response was evaluated in vivo using ELIspot assay. (3) Results: Micelles induced leakage of contents from liposomes via lipid mixing at low pH. Micelles were taken up by dendritic cells mainly via macropinocytosis and delivered ovalbumin (OVA) into the cytosol. After intradermal injection of micelles and OVA, OVA-specific cellular immunity was induced in the spleen. (4) Conclusions: pH-responsive micelles composed of DLPC and deoxycholic acid are promising as enhancers of cytosol delivery of antigens and the induction capability of cellular immunity for the treatment of cancer immunotherapy and infectious diseases.

Development of pH-sensitive Dextran Derivatives with Strong Adjuvant Function and Their Application to Antigen Delivery.

To achieve efficient cancer immunotherapy, the induction of cytotoxic T lymphocyte-based cellular immunity is necessary. In order to induce cellular immunity, antigen carriers that can deliver antigen into cytosol of antigen presenting cells and can activate these cells are required. We previously developed 3-methyl glutarylated dextran (MGlu-Dex) for cytoplasmic delivery of antigen via membrane disruption ability at weakly acidic pH in endosome/lysosomes. MGlu-Dex-modified liposomes delivered model antigens into cytosol of dendritic cells and induced antigen-specific cellular immunity. However, their antitumor effects were not enough to complete the regression of the tumor. In this study, antigen delivery performance of dextran derivatives was improved by the introduction of more hydrophobic spacer groups next to carboxyl groups. 2-Carboxycyclohexane-1-carboxylated dextran (CHex-Dex) was newly synthesized as pH-responsive dextran derivative. CHex-Dex formed stronger hydrophobic domains at extremely weak acidic pH and destabilized lipid membrane more efficiently than MGlu-Dex. CHex-Dex-modified liposomes were taken up by dendritic cells 10 times higher than MGlu-Dex-modified liposomes and delivered model antigen into cytosol. Furthermore, CHex-Dex achieved 600 times higher IL-12 production from dendritic cells than MGlu-Dex. Therefore, CHex-Dex is promising as multifunctional polysaccharide having both cytoplasmic antigen delivery function and strong activation property of dendritic cells for induction of cellular immunity.