Yukihiko Aramaki

Cationic liposomes induce apoptosis through p38 MAP kinase–caspase-8–Bid pathway in macrophage-like RAW264.7 cells

We have demonstrated that cationic liposomes composed of stearylamine (SA‐liposomes) induce apoptosis in a variety of cells, but the mechanism responsible for the cellular death is not clear. In this paper, we investigated the signaling pathways implicated in SA‐liposome‐induced apoptosis in the macrophage‐like cell line RAW264.7. Treatment with SA‐liposomes caused the activation of mitogen‐activated proein kinases (MAPKs), especially p38 and c‐jun N‐terminal kinase, and apoptosis was only inhibited upon the addition of a specific inhibitor for p38. N‐acetylcysteine, a scavenger of reactive oxygen species (ROS), effectively inhibited the activation of p38 and cellular death, indicating that the activation induced by ROS is an initial step in the process of apoptosis triggered by SA‐liposomes. Caspase‐8 was activated by p38, and caspase‐8‐dependent cleavage of Bid was also observed. No down‐regulation of bcl‐2 expression, and no cleavage of Bax protein were observed. Taken together, our results suggest that apoptosis of RAW264.7 by SA‐liposomes was mediated by the MAPK p38 and a caspase‐8‐dependent Bid‐cleavage pathway. Moreover, we found that ROS can contribute intimately to the SA‐liposome‐induced cell death in RAW264.7.

Induction of apoptosis in macrophages by cationic liposomes

The effects of liposomes on apoptosis in macrophages were evaluated from DNA content and DNA fragmentation. Cationic liposomes composed of different kinds of cationic lipids induced apoptosis in mouse splenic macrophages and the macrophage‐like cell line, RAW264.7 cells. Generation of reactive oxygen radicals from macrophages treated with cationic liposomes was detected using flow cytometry, and further apoptosis was inhibited by the addition of oxidant scavenger, N‐acetylcysteine. From these findings, the production of reactive oxygen species may be important in the regulation of apoptosis induced by cationic liposomes.

EFFECTS OF FUSOGENIC AND DNA-BINDING AMPHIPHILIC COMPOUNDS ON THE RECEPTOR-MEDIATED GENE TRANSFER INTO HEPATIC CELLS BY ASIALOFETUIN-LABELED LIPOSOMES

Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer using asialofetuin-labeled liposomes (AF-liposomes) were examined with HepG2 cells and rat hepatocytes in primary culture. AF-liposomes were sufficiently taken up by both types of cells through the asialoglycoprotein receptor-mediated endocytosis. In HepG2 cells, bacterial beta-galactosidase (beta-Gal) gene expression was observed by transfection using AF-liposomes encapsulating plasmid pCMV beta DNA (AF-liposome-pCMV beta). By addition of dioleoylphosphatidylethanolamine (DOPE) to the liposomal lipid composition (AF-liposome(DOPE)-pCMV beta), the transfection efficiency was clearly increased. The effects of DOPE were more conspicuous in the presence of chloroquine in the medium throughout the transfection. When pCMV beta complexed with gramicidin S (pCMV beta (GrS)) was encapsulated (AF-liposome(DOPE)-pCMV beta (GrS) and was transfected to HepG2 cells, an significantly high beta-Gal activity in the cells was observed as compared with that in the cells transfected with AF-liposome(DOPE)-pCMV beta. No effects of GrS were found in the transfection using AF-non-labeled control liposomes. In primary culture of rat hepatocytes, no beta-Gal gene expression was observed even though AF-liposome(DOPE)-pCMV beta was introduced into the cells prepared from adult rats. However, following the transfection with AF-liposome(DOPE)-pCMV beta, the beta-Gal activity was expressed in the cells from immature rats cultured in the medium supplemented with epidermal growth factor and insulin, and the transfection efficiency was 2-fold higher than that transfected with pCMV beta encapsulated in AF-non-labeled control liposomes. By the complex formation of pCMV beta with GrS, the transfection efficiency of AF-liposome(DOPE)-pCMV beta (GrS) increased according to the increase of GrS in the complex. It was shown that AF-liposome(DOPE)-pCMV beta (GrS) did efficiently introduce and express beta-Gal gene in both HepG2 cells and primary hepatocytes in the receptor mediated manner.

Improvement of dermatitis by iontophoretically delivered antisense oligonucleotides for interleukin-10 in NC/Nga mice

IL-10 is overexpressed in skin lesions of atopic dermatitis (AD) patients and believed to be an important factor in the pathogenesis of the disease. Thus the regulation of IL-10 production is a potential solution for immunotherapeutic intervention in AD. We examined the topical delivery of an antisense oligonucleotide for mouse IL-10 (AS6) and the therapeutic effect on the skin lesions of NC/Nga mice, a human AD model. Using an iontophoresis system, about 30% of the applied dose of AS6 penetrated the skin and was distributed in the epidermis and upper dermis. Topically delivered AS6 decreased the levels of mRNA and protein of IL-10 in the lesions of NC/Nga mice, with no effect on IL-4 levels. The dorsal lesions of NC/Nga mice disappeared with repeated topical application of AS6. Topically delivered AS6 showed an inhibitory effect on the production of IL-10 in the skin lesions of NC/Nga mice and had a therapeutic effect on the established dermatitis.

Physicochemical Properties of Liposomes Affecting Apoptosis Induced by Cationic Liposomes in Macrophages

AbstractPurpose. Cationic liposomes are expected to be useful as nonviral vectors for gene delivery. Cationic liposomes showed cytotoxicity, and we proposed that the cytotoxicity is through apoptosis. In this study, we examined the effects of liposomal properties, such as liposomal charge, size, membrane fluidity, and PEG coating, on the induction of apoptosis in the macrophage-like cell line RAW264.7.nMethods. RAW264.7 cells were treated with liposomes, and the induction of apoptosis was evaluated by monitoring the changes in DNA content by flow cytometry. The association of liposomes with cells and the generation of reactive oxygen species (ROS) were also measured by flow cytometry.nResults. The induction of apoptosis of RAW264.7 cells was dependent on the concentrations of stearylamine or cholesterol, a component of cationic liposomes. A significant correlation was observed between the degree of apoptosis and association of cationic liposomes with the cells. Coating the liposomal surface with polyethylene glycol (PEG) decreased the association of cationic liposomes with RAW264.7 cells and reduced the induction of apoptosis. Liposomal size also affected the induction of apoptosis, and larger liposomes showed a higher degree of apoptosis induction. Furthermore, ROS, which were required for the induction of apoptosis by cationic liposomes, were generated in a cholesterol content-dependent manner, and ROS generation was also decreased by PEG coating as the association and the induction of apoptosis were reduced.nConclusions. The degree of apoptosis is related to the extent of association of cationic liposomes with cells and is related to the generation of ROS.

Interferon-γ inductive effect of liposomes as an immunoadjuvant

Abstract Adjuvant effect of liposomes was compared to that of aluminium hydroxide (Alum) using ovalbumin (OVA) as a model antigen, and the difference in adjuvanticity of liposomes from Alum has been evaluated on the basis of cytokine production. Both adjuvants enhanced the IgG levels, but a remarkable difference was observed in the production of IgG subclass; Alum enhanced IgG1 levels, and liposomes enhanced IgG2a, IG2b, and IG3 levels. Further, Alum enhanced antigen-specific IgE levels, whereas liposomes did not. To clarify the difference in adjuvant effect of these adjuvants, secretion of cytokines, especially interferon-γ (IFN-γ) and interleukin-4 (IL-4) which regulate IgE production, was estimated ex vivo . Overnight culture of splenic cells obtained from mice immunized with liposomes encapsulating OVA elicits IFN-γ secretion, but not IL-4 secretion. On the other hand, the production of both cytokines was elevated by the immunization with OVA-Alum complex. The results indicate that the difference of adjuvant activity between liposomes and Alum may come from the difference in the secretion of IL-4 and that, consequently, a different class of antibody response is developed. More importantly, negatively charged liposomes containing phosphatidylserine remarkably promoted IFN-γ secretion compared to neutral liposomes.

Induction of apoptosis in WEHI 231 cells by cationic liposomes.

AbstractPurpose. Liposomes are of considerable interest as drug carriers andimmunoadjuvants. However, few investigators have studied thechanges exerted by liposomes in the cells with which they interact.The purpose of this study was to investigate whether liposomes induceapoptosis in B cells.nMethods. The mouse immature B cell line WEHI 231 cells and mousesplenic B cells were treated with liposomes, and the induction ofapoptosis was evaluated by monitoring changes in DNA content, DNAfragmentation and chromatin condensation by flow cytometry, agarosegel electrophoresis and by morphological investigation.nResults. Cationic liposomes induced apoptosis in WEHI 231 cells, butneutral and anionic liposomes did not. A contact time of 30 minbetween WEHI 231 cells and cationic liposomes was sufficient toinduce apoptosis, and 80% of the cells showed hypodiploid DNAcontent. Apoptosis induced by cationic liposomes composed ofstearylamine was inhibited by addition of the oxidant scavenger,N-acetyl-cysteine.nConclusions. Cationic liposomes induced apoptosis in WEHI 231 cells,and the production of reactive oxygen species is important in theregulation of apoptosis induced by cationic liposomes. It is well knownthat cationic liposomes show cytotoxicity, and apoptosis may be oneof the causes of this toxicity.

Uptake of phosphatidylserine liposomes by rat Peyer's patches following intraluminal administration

Uptake of the nonabsorbable marker 6-carboxyfluorescein was investigated both free and encapsulated in liposomes as a function of their surface charge and hydrodynamic diameter in rat Peyers patch and nonpatch tissue. Significant uptake of the marker occurred only when encapsulated in liposomes consisting of at least 25 mol% phosphatidylserine and was highest in Peyers patches. 6-Carboxyfluorescein encapsulated in liposomes equal to or greater than 374 nm was preferentially taken up by Peyers patches. There was a trend to higher uptake in lower intestinal segments. These findings were supported by fluorescence microscopic observations. Uptake by Peyers patches was specific for negatively charged liposomes as judged from competition studies.

Drug release from pH-response polyvinylacetal diethylaminoacetate hydrogel, and application to nasal delivery

Abstract Nasal formulations of polyvinylacetal diethylaminoacetate (AEA) were prepared and the effect of AEA concentration on drug release was evaluated in in vitro and in vivo experiments. The profiles of release of drug from dialysis tubes had both a rapid and a slow phase, and had an inflection point, at which AEA hydrogel formation appeared to occur. The higher the AEA concentration, the lower the rate of drug release observed. The apparent disappearance rate constant ( k app ) of drug was determined by the deposit method, which estimates changes in the amount of residual drug in the nasal cavity with the lavage technique following administration. Drug k app values decreased with increase in AEA concentration. Hydrogel formation on mucous membranes was also visually confirmed in rat nasal cavity. AEA preparations which facilitate instillation into the nose but which form hydrogel on the mucous membrane are potentially useful for controlled-release nasal delivery systems.

Involvement of phosphatidylinositol-3-kinase and ERK pathways in the production of TGF-β1 by macrophages treated with liposomes composed of phosphatidylserine

We explored the involvement of phosphatidylinositol 3‐kinase (PI3K) and ERK pathways in the production of TGF‐β1 by macrophages treated with liposomes composed of phosphatidylserine (PS‐liposomes). PS‐liposomes activated Akt, downstream of the PI3K signal cascade, and ERK which led to the expression of TGF‐β1. PI3K inhibitors, LY294002 and wortmannin, inhibited the activation of Akt and ERK following the treatment with PS‐liposomes. These inhibitors also suppressed the production of TGF‐β1. Furthermore, PS‐liposomes activated macrophages to induce TGF‐β1 expression through PS‐specific receptors. These findings suggested that a PI3K‐ERK signaling pathway via the PS‐receptor is intimately involved in the production of TGF‐β1 which regulates macrophage functions.