Yoshikazu Inoh

The ratio of unsaturated fatty acids in biosurfactants affects the efficiency of gene transfection.

An unsaturated hydrocarbon chain in phospholipid was reported to affect a phase transition and a fusogenic activity after mixing membranes, and consequently to achieve a high DNA transfection efficiency. We previously showed that a biosurfactant mannosylerythritol lipid-A (MEL-A) enhances the gene transfection efficiency of cationic liposomes. Here, we have studied the effects of unsaturated fatty acid ratio of MEL-A on the physicochemical properties and gene delivery into cells of cationic liposomes using MEL-A with three different unsaturated fatty acid ratios (9.1%, 21.5%, and 46.3%). The gene transfer efficiency of cationic liposomes containing MEL-A (21.5%) was much higher than that of those containing MEL-A (9.1%) and MEL-A (46.3%). MEL-A (21.5%)-containing cationic liposomes induced highly efficient membrane fusion after addition of anionic liposomes and led to subsequent DNA release. Imaging analysis revealed that MEL-A (21.5%)-containing liposomes fused with the plasma membrane and delivered DNA into the nucleus of NIH-3T3 cells, MEL-A (46.3%)-containing liposomes fused with the plasma membrane did not deliver DNA into the nucleus, and MEL-A (9.1%)-containing liposomes neither fused with the plasma membrane nor delivered DNA into the nucleus. Thus, it is understandable that the unsaturated fatty acid ratio of MEL-A strongly influences the gene transfection efficiency of cationic liposomes.

Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes.

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.

Nano vectors with a biosurfactant for gene transfection and drug delivery

Nano vectors are useful tools for delivery of foreign DNA and oligonucleotides into mammalian cells in gene transfection, drug delivery, and gene therapy. In experiments with nano vectors the cationic liposomes have been widely used for their high transfection efficiency and low toxicity. Recently, we have found that nano vectors containing a biosurfactant, which is a surface-active compound produced by microorganisms growing on water-insoluble substrates, increased the efficiency in gene transfection dramatically in vitro and in vivo. This was due to a new pathway of gene delivery into the target cells via the fusion with the plasma membranes. In this paper, we will introduce this interesting nano vector, as we believe it will become a new operating technique with great potential.

Synergistic effect of a biosurfactant and protamine on gene transfection efficiency.

Several barriers need to be overcome to ensure successful gene transfection, including passing of the foreign gene through the plasma membrane, escape of this material from lysosomal degradation, and its translocation into the nucleus. We previously showed that the biosurfactant mannosylerythritol lipid-A (MEL-A) enhanced the efficiency of gene transfection mediated by cationic liposomes by facilitating rapid delivery of foreign genes into target cells through membrane fusion between liposomes and the plasma membrane. Moreover, using MEL-A-containing cationic liposomes, the foreign gene was efficiently delivered into the nucleus because it was released directly into the cytosol and thus escaped lysosomal degradation. Here we investigated the effect of pre-condensation of plasmid DNA by a cationic polymer, protamine, on gene transfection. We found that the efficiency of pre-condensed DNA transfection mediated by MEL-A-containing OH liposomes was >10 times higher than that of non-condensed DNA transfection. In contrast, the efficiency of pre-condensed DNA transfection mediated by OH liposomes was only 1.5 times higher than that of non-condensed DNA transfection. MEL-A did not influence plasmid DNA encapsulation by cationic liposomes, but it greatly accelerated the nuclear delivery of pre-condensed plasmid DNA. Our findings indicate that MEL-A and protamine synergistically accelerate the nuclear delivery of foreign gene and consequently promote gene transfection efficiency.

Gene transfection efficiency into dendritic cells is influenced by the size of cationic liposomes/DNA complexes

&NA; Cationic liposomes have attracted recent attention as DNA vaccine carriers that can target dendritic cells (DCs). In general, cationic liposome/DNA complexes (lipoplexes) are taken up by various cells via clathrin‐mediated endocytosis, caveolae‐mediated endocytosis, macropinocytosis, or phagocytosis, with the mode of endocytosis determining further intracellular trafficking pathways. Moreover, the physicochemical properties of cationic lipoplexes, including lipid composition, shape, size, and charge, influence transfection efficiency, affecting uptake and subsequent intracellular pathways. To develop cationic liposomes as potential DNA vaccine carriers, the objective of this study was to study the effect of lipoplex size on DNA transfection efficiency in DCs. We explored the size‐dependent endocytosis pathway and the intracellular trafficking of cationic lipoplexes using bone marrow derived dendritic cells (BMDCs). Our results indicated that small‐sized lipoplexes (approximately 270 nm diameter) were taken up by BMDCs via caveolae‐mediated endocytosis, which led to a non‐degradative pathway, whereas larger‐sized lipoplexes (approximately 500 nm diameter) were taken up by BMDCs via clathrin‐mediated endocytosis and micropinocytosis, which led to a lysosomal degradation pathway. These findings suggest that, by regulating the size of lipoplexes, it may be possible to develop cationic liposomes as DNA vaccine therapies for targeting DCs. Graphical Abstract Figure. No caption available.

Impaired expression of the mitochondrial calcium uniporter suppresses mast cell degranulation.

Calcium ion (Ca2+) uptake into the mitochondrial matrix influences ATP production, Ca2+ homeostasis, and apoptosis regulation. Ca2+ uptake across the ion-impermeable inner mitochondrial membrane is mediated by the mitochondrial Ca2+ uniporter (MCU) complex. The MCU complex forms a pore structure composed of several proteins. MCU is a Ca2+-selective channel in the inner-mitochondrial membrane that allows electrophoretic Ca2+ entry into the matrix. Mitochondrial Ca2+ uptake 1 (MICU1) functions as a Ca2+-sensing regulator of the MCU complex. Previously, by microscopic analysis at the single-cell level, we found that during mast cell activation, mitochondria capture cytosolic Ca2+ in two steps. Consequently, mitochondrial Ca2+ uptake likely plays a role in cellular function through cytosolic Ca2+ buffering. Here, we investigate the role of MCU and MICU1 in mitochondrial Ca2+ uptake and mast cell degranulation using MCU- and MICU1-knockdown (KD) mast cells. Whereas MCU- and MICU1-KD mast cells show normal proliferation rates and mitochondrial membrane potential, they exhibit slow and reduced cytosolic and mitochondrial Ca2+ elevation after antigen stimulation. Moreover, β-hexosaminidase release induced by antigen was significantly suppressed in MCU-KD cells but not MICU1-KD cells. This suggests that both MCU and MICU1 are involved in mitochondrial Ca2+ uptake in mast cells, while MCU plays a role in mast cell degranulation.

Substance P plays an important role in cell adhesion molecule 1-mediated nerve–pancreatic islet α cell interaction

Autonomic neurons innervate pancreatic islets of Langerhans and maintain blood glucose homeostasis by regulating hormone levels. We previously showed that cell adhesion molecule 1 (CADM1) mediated the attachment and interaction between nerves and aggregated pancreatic islet α cells. In this study, we cocultured αTC6 cells, a murine α cell line, with mouse superior cervical ganglion (SCG) neurons. The oscillation of intracellular Ca(2+) concentration ([Ca(2+)]i) was observed in 27% and 14% of αTC6 and CADM1-knockdown αTC6 cells (αTC6(siRNA-CADM1) cells) in aggregates, respectively, within 1min after specific SCG nerve stimulation with scorpion venom. In αTC6(siRNA-CADM1) cells, the responding rate during 3min after SCG nerve stimulation significantly increased compared with that within 1min, whereas the increase in the responding rate was not significantly different in αTC6 cells. This indicated that the response of αTC6 cells according to nerve stimulation occurred more rapidly and effectively than that of αTC6(siRNA-CADM1) cells, suggesting CADM1 involvement in promoting the interaction between nerves and α cells and among α cells. In addition, because we found that neurokinin (NK)-1 receptors, which are neuropeptide substance P receptors, were expressed to a similar extent by both cells, we investigated the effect of substance P on nerve-α cell interaction. Pretreatment with CP99,994 (0.1μg/ml), an NK-1 receptor antagonist, reduced the responding rate of both cells, suggesting that substance P released from stimulated neurites was a mediator to activate αTC6 cells. In addition, α cells that were attached to neurites in a CADM1-mediated manner appeared to respond effectively to neurite activation via substance P/NK-1 receptors.

Inhibitory effects of a cationic liposome on allergic reaction mediated by mast cell activation

Several studies have shown that cationic liposomes exert immunomodulatory effects with low immunogenicity and toxicity, and offer advantages such as easy preparation and targeting. Cationic liposomes not only transport DNA to immune cells but also enhance the function of antigen presenting cells such as dendritic cells and macrophages. Here, we investigated the effect of a particular cationic liposome on mast cell function during allergic reaction. We found that the cationic liposomes bound to the mast cell surface suppressed degranulation induced by cross-linking of high affinity immunoglobulin E receptors in a time- and dose-dependent manner. The suppression of degranulation was mediated by impairment of the sustained level of intracellular Ca(2+) concentration ([Ca(2+)]i) derived from the inhibition of store-operated Ca(2+) entry. The decrease in sustained elevation of [Ca(2+)]i led to the suppression of phosphorylation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins such as SNAP-23, syntaxin-4, which are necessary for membrane fusion between secretory granules and the plasma membrane during degranulation. Furthermore, the cationic liposomes suppressed vascular permeability elevation induced by mast cell activation in mice. These results showed that cationic liposomes possess the novel property of inhibiting mast cell activation, suggesting the possibility of developing cationic liposomes as anti-allergic effectors.

Phosphorylation of syntaxin‐3 at Thr 14 negatively regulates exocytosis in RBL‐2H3 mast cells

Recent studies have revealed that soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins interact with each other, forming a SNARE complex that induces exocytosis in mast cells. Previously, we reported that syntaxin‐3, a SNARE protein, regulates mast cell exocytosis and is constantly phosphorylated. In this study, we tried to identify the amino acid residue that is phosphorylated in mast cells, and to elucidate the regulatory mechanism of exocytosis by phosphorylation in syntaxin‐3. We found that Thr 14 of syntaxin‐3 was a phosphorylation site in mast cells. In addition, the overexpression of a constitutively dephosphorylated syntaxin‐3 (T14A) mutant enhanced mast cell exocytosis. We also showed that the phosphomimetic mutation of syntaxin‐3 at Thr 14 (T14E) induced structural changes in syntaxin‐3, and this mutation inhibited binding of syntaxin‐3 to Munc18‐2. These results suggest that phosphorylated syntaxin‐3 at Thr 14 negatively regulates mast cell exocytosis by impairing the interaction between syntaxin‐3 and Munc18‐2.

New Transfection Agents Based on Liposomes Containing Biosurfactant MEL-A

Nano vectors are useful tools to deliver foreign DNAs, oligonucleotides, and small interfering double-stranded RNAs (siRNAs) into mammalian cells with gene transfection and gene regulation. In such experiments we have found the liposomes with a biosurfacant mannosylerythriol lipid (MEL-A) are useful because of their high transfer efficiency, and their unique mechanism to transfer genes to target cells with the lowest toxicity. In the present review we will describe our current work, which may contribute to the great advance of gene transfer to target cells and gene regulations. For more than two decades, the liposome technologies have changed dramatically and various methods have been proposed in the fields of biochemistry, cell biology, biotechnology, and so on. In addition, they were towards to pharmaceutics and clinical applications. The liposome technologies were expected to use gene therapy, however, they have not reached a requested goal as of yet. In the present paper we would like to present an approach using a biosurfactant, MEL-A, which is a surface-active compound produced by microorganisms growing on water-insoluble substrates and increases efficiency in gene transfection. The present work shows new transfection agents based on liposomes containing biosurfactant MEL-A.