Toshifumi Hara

Stability of liposomes in vitro and their uptake by rat Peyer's patches following oral administration

To evaluate the usefulness of liposomes as a carrier for the targeted delivery of antigens to gut-associated lymphoid tissue, liposomal stability and uptake by rat Peyers patches were investigated. Liposomes composed of distearoylphosphatidylcholine, phosphatidylserine, and cholesterol (DSPC-liposome), or dipalmitoylphosphatidylcholine, phosphatidylserine, and cholesterol were stable in acidic solution (pH 2.0), diluted bile, and pancreatin solution. Following the oral administration of liposomes to rats, rhodamine B-PE incorporated in the lipid phase of DSPC-liposomes was preferentially taken up by Peyers patches in the lower ileum. The uptake of rhodamine B-PE from DSPC-liposomes larger than 374 nm in mean diameter was high. Orally administered DSPC-liposomes of a large diameter thus appear to serve effectively as a vehicle for delivering antigens to Peyers patches.

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.

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.

Enhancement of systemic and mucosal immune responses following oral administration of liposomes

The effects of liposomes on the systemic and mucosal immune response were investigated using bovine serum albumin (BSA) in BALB/c mice. Following three oral administrations of varied formulations at 1-week intervals, serum BSA-specific IgG levels were increased significantly by BSA encapsulated in liposomes and moderately by a mixture of liposomes and BSA. Serum and salivary BSA-specific IgA levels were elevated by BSA-encapsulating liposomes only. Liposomes thus activate not only the systemic immune response but also the mucosal immune response following their oral administration. However, no increase in salivary IgA levels was observed by intraperitoneal or subcutaneous injection of BSA-encapsulating liposomes. The production of IgA is closely related to the oral administration of liposomes encapsulating antigens. Liposomes thus function as carriers of oral vaccines against various infections of the mucosal surface.

Specific uptake of asialofetuin-tacked liposomes encapsulating interferon-γ by human hepatoma cells and its inhibitory effect on hepatitis B virus replication

Asialofetuin-tacked liposomes (AF-liposomes) encapsulating interferon (IFN)-gamma were bound and internalized into a human hepatoma cell line, HepG2 cells, selectively through asialoglycoprotein receptor, but not non-tacked liposomes (N-liposomes). AF-liposomal IFN-gamma was more effective for inhibition of viral DNA replication in hepatitis B virus (HBV)-producing clone from HepG2 cells transfected with HBV-DNA than N-liposomal IFN-gamma. AF-liposomes may increase the therapeutic potential of IFN-gamma through asialoglycoprotein receptor in treating HBV-infected hepatocytes.

Recognition of charged liposomes by rat peritoneal and splenic macrophages: effects of fibronectin on the uptake of charged liposomes

Abstract Different recognition systems for charged liposomes were found in rat peritoneal and splenic macrophages. In peritoneal macrophages, significant uptake was observed in negatively charged liposomes, depending on the phosphatidylserine concentration, and receptor-mediated uptake of negatively charged liposomes was suggested. In contrast, splenic macrophages took up positively charged liposomes significantly, depending on the stearylamine concentration, and participation of receptor-mediated uptake process was also suggested. Fibronectin purified from rat plasma bound to saturation to positively charged liposomes preferentially. The addition of fibronectin caused significant increase in the uptake of liposomes containing stearylamine by splenic macrophages. The uptake of liposomes by peritoneal macrophages was not enhanced by fibronectin irrespective of liposomal charge. Rat plasma fibronectin may thus be concluded to function as an opsonin of positively charged liposomes for rat splenic macrophages.

Preparation of asialofetuin-labeled liposomes with encapsulated human interferon-gamma and their uptake by isolated rat hepatocytes.

The selective delivery of human recombinant interferon (IFN)-γ to isolated rat hepatocytes was studied with asialofetuin (AF)-labeled liposomes. AF-liposomes containing buffer solution were initially prepared by the detergent removal method, and IFN-γ was subsequently encapsulated by the freeze-thawing method without loss of activity. Virtually no free [32P]IFN-γ was internalized into isolated rat hepatocytes, whereas AF-liposomes containing [32P]IFN-γ were taken up to a significant degree. Liposomal binding to the hepatocytes (estimated at 4°C) was one-fifth of the uptake (estimated at 37°C). Since the uptake was inhibited by the addition of free AF, AF-liposomes may be taken up by the action of galactose-binding protein on the hepatocytic cell surface. The liposome preparation method reported in this paper provides a useful means for the encapsulation of unstable macromolecules into AF-liposomes. AF-liposomes were found effectively to carry IFN-γ into hepatocytes in vitro.

Characteristics of the binding of asialofetuin-labeled liposomes to isolated rat hepatocytes

The binding of asialofetuin(AF)-labeled liposomes (AF-liposomes) to isolated rat hepatocytes was examined at 4°C. AF-liposomes consisting of phosphatidylcholine, phosphatidic acid (PA) and cholesterol (molar ratio 6: 1: 3) rapidly bound to hepatocytes. A notable contribution of AF was demonstrated by the fact that the binding was inhibited by AF and dissociated by EDTA. An increase in the PA content of AF-liposomes enhanced the extent of their binding, whereas the extent of AF-mediated binding remained the same irrespective of the PA content. There were two classes of binding sites at which AF-liposomes bound to hepatocytes: a high-affinity site with an association constant of 6.45 × 109 M−1 and a low-affinity site with an association constant of 4.50 × 107 M−1, this value being compatible with that of control liposomes. Phosphatidylserine- or phosphatidylglycerol-containing liposomes bound to hepatocytes as did liposomes containing PA. The binding of stearylamine-containing liposomes or liposomes without charged lipid, however, was quite low. Thus, it is evident that AF-liposomes bind to hepatocytes through the high-affinity interaction between AF and asialoglycoprotein receptors and the low-affinity interaction that occurs as a result of liposomal negative charge.

Liposomal Induction of a Heat-stable Macrophage Priming Factor to Induce Nitric Oxide in Response to LPS

AbstractPurpose. The effects of liposomes on nitric oxide (NO) production from mouse peritoneal macrophages following intraperitoneal injection of liposomes were investigated. Methods. Mouse peritoneal macrophages were collected following intraperitoneal injection of liposomes and cultured with and without lipopolysaccharide (LPS). Peritoneal washing fluid was also collected from the mice injected with liposomes. NO production was evaluated by measuring the concentration of nitrite in the macrophage culture supernatant by Griess reagent. Results. NO production stimulated by LPS was observed in peritoneal macrophages obtained from the liposome-treated mice, but liposomes did not activate macrophages directly to induce NO in response to LPS. NO production was higher in the liposomes composed of phospha-tidylcholine than that of negatively charged liposomes composed of phosphatidylserine. Peritoneal washing fluid obtained from mice injected with liposomes has a capacity to induce NO production in the macrophages from naive mice. This capacity was not diminished by heat-treatment at 100°C for 5 min. Conclusions. Peritoneal macrophages were activated to produce NO in response to LPS following intraperitoneal injection of liposomes. They did not activate macrophages directly, and the induction of heat-stable macrophage priming factor, but not cytokines, is suggested.

Selective cytotoxicity of EFG labeled liposomes encapsulating bleomycin for A431 cells.

EGF-labeled reverse phase evaporation vesicles (EGF-REV) encapsulating bleomycin (BLM) have been prepared and their selective uptake and cytotoxic activity have been evaluated using A431 and Swiss/3T3 cells which are over-expressing EGF-receptors on cell surface and no EGF-receptor, respectively. EGF has been bound to REV surface as active form, and EGF-REV has been taken up selectively by A431 cells via EGF-receptors, but not by Swiss/3T3 cells. Cytotoxic activity of EGF-REV encapsulating BLM (EGF-REV-BLM) for A431 cells was superior to that of normal liposomes containing BLM, and this effect was dependent on the treatment time. However, this cytotoxic activity of EGF-REV-BLM could not be observed in Swiss/3T3 cells, These findings suggested that EGF-REV is a useful drug carrier for squamous carcinoma which are over-expressing EGF-receptors on cell surface.