Selective uptake by cancer cells of liposomes coated with polysaccharides bearing 1-aminolactose.

We investigated the selective uptake of liposomes chemically modified by polysaccharides-cholesterol derivatives with 1-aminolactose (lactose) in two human hepatoma cell lines (HUH7 and Alexander), a human colon cancer cell line (FCC) and a human lung cancer cell line (KNS). The uptakes of the labeled liposomes alone (conventional liposomes), those with cholesterol pullulan (CHP) and with lactose (lactose CHP) were compared in four cancer cells and normal rat hepatocytes after 3 hours of incubation. The radioactivities of the lactose CHP were 4.4, 4, 3.4 and 4.4 times greater than those of CHP in HuH7, Alexander, FCC and KNS cells, respectively, after 3 hours of incubation. All the above differences were statistically significant (p < 0.01). No statistically significant differences were seen in the case of hepatocytes. Thus, cancer cells have a common affinity with lactose CHP liposomes, however, these mechanisms appear to have no connection with the galactose-specific asialoglycoprotein receptors of hepatocytes.

Development of a cancer vaccine: peptides, proteins, and DNA.

Genetic changes leading to protooncogene activation qualitatively and/or quantitatively alter their gene products and are exclusively or largely restricted to transforming cells and their precursors. The overexpression of HER2 is among those changes and is often detected in adenocarcinomas such as breast, ovarian, lung, and gastric cancer. This provides a rationale for exploring the possibility that HER2 is a target of host immune responses against cancer cells. We have recently demonstrated that HER2 can be a target for tumor-rejecting immune responses against syngeneic murine HER2+ tumor cells. We defined two different peptides, HER2p63-71 and HER2p780-788, with a Kd anchor motif that can induce CD8+ cytotoxic T lymphocytes (CTLs). The growth of HER2+ syngeneic tumors was suppressed in mice immunized with HER2p63-71 or p780-788. Since murine Kd and human HLA-A24 share a similar anchor motif for peptides, HER2p63 71 and HER2p780-788 were examined for induction of CTLs in HLA-A24+ individuals. CD8+ CTL clones specific for these peptides were established and they lysed HER2+ tumor cells in a human leukocyte antigen (HLA)-A24-restricted manner. To elicit specific CD8+ T cell immune responses against cancer, the development of efficient devices to deliver tumor antigen peptides to the major histocompatibility complex (MHC) class I pathway constitutes a central issue. We have developed a novel formula of hydrophobized polysaccharide nanoparticles which can deliver a HER2 oncoprotein containing an epitope peptide to the MHC class I pathway. We designed a simple protein delivery system: cholesteryl group-bearing polysaccharides, mannan or pullulan (CHM or CHP, respectively), complexed with the truncated HER2 protein containing the 147 N-terminal amino acids. These complexes were able to induce CD8+ CTLs against HER2+ tumors. CTLs were MHC class I restricted and specifically recognized HER2p63-71, a part of a truncated HER2 protein used as an immunogen. The complete rejection of tumors also occurred when CHM-HER2 was applied early after tumor implantation. In the effector phase of in vivo tumor rejection, CD8+ T cells played a major role. The results suggest that this unique hydrophobized polysaccharide may help soluble proteins to induce cellular immunity. Such a novel vaccine may be of potential benefit in cancer prevention and cancer therapy.

Utility of liposomes coated with polysaccharide bearing 1-amino-lactose as targeting chemotherapy for AH66 hepatoma cells.

The cell recognition element is very important for drug delivery systems. We synthesized cholesteryl pullulan (CHP) bearing 1-aminolactose (1-AL) and introduced a saccharide, cholesteryl pullulan bearing 1-aminolactose (1-AL/CHP), to an outer layer of the conventional liposome as a cell recognition element. Lectin recognized the beta-galactose by aggregation of 1-AL/CHP coated liposome (1-AL/CHP liposome). The uptake of this liposome to AH66 rat hepatoma cells was greater than in liposomes without 1-aminolactose in vitro. Furthermore, 1-AL/CHP liposomal adriamycin showed a stronger antitumor effect in comparison with other types of liposomal adriamycin in vitro. When in vivo tumor-targeting efficacy was investigated in AH66 tumor transplanted mice using 3H-liposome, the tumor/serum radioactivity ratio in mice injected with 1-AL/CHP liposome was higher than that of mice injected with other liposomes. These observations suggest that 1-AL is effective as a cell recognition element. As a result, 1-AL/CHP liposome is considered to be a good carrier of anticancer drugs for the active targeting of tumor cells.

Activation of human T lymphocytes by ganglioside-containing liposomes.

We investigated the in vitro stimulatory effect of ganglioside (GM3, GD1a, GD1b, GT1b, or GQ1b)-containing liposomes on human immune cells. The effect of ganglioside-containing liposomes on the concentration of cytoplasmic free calcium ions ([Ca2+]i) in human immunocytes was examined using the confocal laser fluorescence microscopic method. The GD1a- and GT1b-containing liposomes significantly increased [Ca2+]i of human T lymphocytes compared with the GM3-, GD1b- and GQ1b-containing ones. The response of CD8+ and CD4+ cells was significantly higher than that of CD20+ cells. Our results show that the increase in [Ca2+]i may be caused by not the number of sialic acids contained in the gangliosides but the conformation of the sialic acid moiety to protrude exteriorly from the liposomal membrane surface, and that a sort of receptor recognizing the sialic acid moiety exists on human T lymphocytes (both CD8+ and CD4+ cells), which may be involved in the activation of the cells. The present results are almost the same as those obtained for the rat T lymphocyte system previously reported. This clearly confirms that a sort of ganglioside surely stimulates T lymphocytes directly, which is not species-specific but conserved in humans and rats among animal species.

Molecular chaperone-like activity of hydrogel nanoparticles of hydrophobized pullulan: thermal stabilization with refolding of carbonic anhydrase B.

We have been studying the formation of hydrogel nanoparticles by the self-aggregation of hydrophobized polysaccharide and the effective complexation between these nanoparticles as a host and various globular soluble proteins as a guest. This paper describes a new finding that refolding of the heat-denatured enzyme effectively occurs with the nanoparticles and beta-cyclodextrin according to a mechanism similar to that of a molecular chaperone. In particular, the irreversible aggregation of carbonic anhydrase B (CAB) upon heating was completely prevented by complexation between the heat-denatured enzyme and hydrogel nanoparticles formed by the self-aggregation of cholesteryl group-bearing pullulan (CHP). The complexed CAB was released by dissociation of the self-aggregate upon the addition of beta-cyclodextrin. The released CAB refolded to the native form, and almost 100% recovery of the activity was achieved. The thermal stability of CAB was drastically improved by capture of the unfolded form which was then released to undergo refolding.

Induction of acetylcholinesterase release from erythrocytes in the presence of liposomes.

When human erythrocytes are incubated with liposomes, the release of acetylcholinesterase (AChE) occurs following an induction period [Cook et al. (1980) Biochemistry 19, 4601-4607]. However, the mechanism of the induction has not been elucidated. We examined the relationships among the lipid transfer from liposomes to erythrocytes, the morphological change of erythrocytes, the fluidity of the erythrocyte membrane and the start of AChE release. The AChE release into the liposomes and into shed-vesicle fractions started simultaneously after an induction period. The morphological index (MI) of erythrocytes was approximately 2.8 at the beginning of the release, regardless of the induction period. AChE was not released from the erythrocytes of index 2.8 even in the presence of liposomes if the MI remained at 2.8. Therefore, for the release, erythrocytes needed a further increase of the MI from 2.8. As the rate of lipid transfer increased, the induction period became shorter. No significant lipid release from erythrocytes was detected during the induction period. The initiation of the AChE release was not simply affected by the change in the membrane fluidity of erythrocytes upon interaction with liposomes. These results first demonstrate that AChE release into the shed-vesicle and liposome fractions is triggered by a further increase of the MI from 2.8, which is induced by lipid transfer from liposomes to erythrocytes.

Bone marrow-derived dendritic cells incorporate and process hydrophobized polysaccharide/oncoprotein complex as antigen presenting cells.

We have previously shown that a novel hydrophobized polysaccharide/oncoprotein complex vaccine can induce immune responses against the HER2/neu/c-erbB2 (HER2) expressing tumors. Bone marrow-derived dendritic cells (DCs), as antigen presenting cells (APCs), are the first candidates for presentation of tumor antigens. The aim of this study was to see whether DCs are able to elicit antigen specific host immune responses by stimulating the proliferation of T cells after exposure to cholesteryl group bearing pullulan (CHP) and HER2 protein complex. Vaccination by CHP-HER2 complex was as effective as cholesteryl group bearing mannan (CHM) and HER2 complex on which we reported previously. Immunization of mice with HER2 expressing CMS17HE tumor cells generated both CD4+ T cells and CD8+ T cells reactive with CHP-HER2 complex pretreated DCs. In addition, immunization with either CHP-HER2 complex or HER2 protein alone could also generate both CD4+ T cells and CD8+ T cells specifically reactive with CHP-HER2 complex pretreated DCs. The complete rejection of tumors occurred when immunization with CHP-HER2 complex pretreated DCs was started 10 days after tumor inoculation. Therefore, bone marrow-derived DCs pretreated with hydrophobized polysaccharide/oncoprotein complex are a powerful tool for enhancing the effectiveness of oncoprotein for anti-tumor vaccination, opening new options for immune cell therapy.

Self-assembled hydrogel nanoparticle of cholesterol-bearing pullulan as a carrier of protein drugs: complexation and stabilization of insulin.

Insulin (Ins) spontaneously and easily complexed with the hydrogel nanoparticle of hydrophobized cholesterol-bearing pullulan (CHP) in water. The complexed nanoparticles (diameter 20-30 nm) thus obtained formed a very stable colloid. The thermal denaturation and subsequent aggregation of Ins were effectively suppressed upon complexation. The complexed Ins was significantly protected from enzymatic degradation. Spontaneous dissociation of Ins from the complex was barely observed, except in the presence of bovine serum albumin. The original physiological activity of complexed Ins was preserved in vivo after i.v. injection.

A novel hydrophobized polysaccharide/oncoprotein complex vaccine induces in vitro and in vivo cellular and humoral immune responses against HER2-expressing murine sarcomas.

To elicit specific cellular immune responses against cancer, the development of efficient devices to deliver tumor antigen peptides to the MHC class I pathway constitutes a central issue. We report here a novel formula of hydrophobized polysaccharide nanoparticles, which can deliver a HER2 oncoprotein containing an epitope peptide to the MHC class I pathway. A protein consisting of the 147 amino-terminal amino acids of oncogene erbB-2/neu/HER2 (HER2) was complexed with two kinds of hydrophobized polysaccharides, cholesteryl group-bearing mannan (CHM) and cholesteryl group-bearing pullulan (CHP), to form nanoparticles (CHM-HER2 and CHP-HER2). CHM-HER2 and CHP-HER2 were able to induce CD3+/CD8+ CTLs against HER2-transfected syngeneic fibrosarcoma cell lines. In contrast, the oncoprotein alone failed to do so. These CTLs were Kd-restricted and specifically recognized a peptide (position 63-71) that was a part of a truncated HER2 protein used as an immunogen. In addition, vaccination by CHM-HER2 complexes led to a strongly enhanced production of IgG antibodies against HER2, whereas vaccination with HER2 proteins alone resulted in a production of antibodies at a marginal level. Mice immunized with CHM-HER2 or CHP-HER2 before tumor challenge successfully rejected HER2-transfected tumors. The complete rejection of tumors also occurred when CHM-HER2 was applied not later than 3 days after tumor implantation. In the effector phase of in vivo tumor rejection, CD8+ T cells played a major role. The results suggest that a sort of hydrophobized polysaccharide may help soluble proteins to induce cellular immunity as well enhance humoral immunity; hence, such a novel vaccine may be of potential benefit to cancer prevention and cancer therapy.

Antitumor effect of polysaccharide coated liposomal adriamycin on AH66 hepatoma in nude mice.

Drug delivery systems play a crucial role in cancer chemotherapy, not only in the achievement of sufficient tumoricidal effect but also in minimizing systemic side effects. We investigated the effect of liposomal adriamycin with tumor recognition molecule, 1-aminolactose (1-AL), on AH66 hepatoma transplanted into nude mice. Adriamycin (ADM) was encapsulated in liposome coating with cholesterol pullulan (CHP) to increase the stability in the blood stream. 1-AL was assembled to the outer layer of CHP-coated liposomal ADM as a tumor recognition molecule. In an in vivo therapeutic study. 1-AL/CHP-coated liposomal ADM restrained tumor growth more when compared with CHP-coated liposomal ADM. Thus, 1-AL/CHP-coated liposome seems to be a carrier of ADM to tumor cells.

A novel monoclonal antibody that recognizes apical membrane of frog taste cells.

We established a hybridoma clone 1N1 that produced a monoclonal antibody to stain the apical portion of frog taste cells, by directly immunizing taste discs of the bullfrog (Rana catesbeiana) without any dispersion procedure of the taste organ. The antibody stained discrete regions on the surface of the taste discs, but did not stain the epithelium sheet of the tongue devoid of taste discs. The antibody stained approximately 93% of the taste discs tested (172/184) derived from nine frogs, showing that distribution of the antigen was common to most of the taste discs. The following observations strongly suggested that the antibody recognized a certain antigen on the apical membrane of the taste cells. (i) The antibody selectively stained cross points of intermucus areas on the surface of the taste disc. Neither the mucus cells nor the wing cells that mainly cover the surface were stained with the antibody. (ii) Dispersed taste cells were prepared by calcium ion chelating and subsequently by collagenase treatment to avoid digestion of the antigen. The antibody stained the apical end of the taste cells.

Liposome-induced release of cell membrane proteins from intact tissue epithelium.

During extraction and purification, membrane proteins very often undergo denaturation and deactivation. To overcome this problem, the authors have tried to establish a better methodology to make the study at in vivo tissue level, not at the isolated cellular level, possible and easier. This is in vivo direct exposure of animal tissue to the liposome that contains an artificial boundary lipid (D14DPC, 1,2-dimyristamido-1,2,-deoxyphosphatidylcholine). Bullfrog and rat tongues were used. To confirm the reasonableness of this methodology, several different techniques were adopted; the nerve response study, gel electrophoretic analysis, quartz crystal microbalance (QCM) measurement and the affinity gelchromatography. When the tongue was exposed to the D14DPC-containing DMPC liposome, a significant amount of membrane protein was found in the recovered liposome (this was the production of proteoliposome). The nerve response in the neurophysiological measurement to several taste stimuli, such as L-alanine, L-leucine, sucrose and quinine hydrochloride significantly decreased when the tongue was exposed to the same liposome. These phenomena were common to both bullfrog and rat tongues. The nerve response to the stimulation with L-alanine was the most remarkably affected in the liposomal treatment. Therefore, the L-alanine-binding protein was focused upon to confirm the reasonableness of the QCM measurement and the affinity gelchromatography. The D14DPC-containing proteoliposome always showed significant binding to both the L-alanine affinity gel and the L-alanine-conjugated QCM. The results revealed that membrane proteins can be directly and effectively released, even from intact animal tissue epithelium, using the artificial boundary lipid-containing liposome.

Fusion between Jurkat cell and PEO-lipid modified liposome.

Direct fusion between Jurkat cell and a liposome modified with poly(ethylene oxide)-bearing lipid (PEO-lipid) was examined using diphtheria toxin fragment A (DTA) as the probe. Only the DTA-loaded liposome modified with PEO-lipid(n = 32) (n is the number of ethylene oxide units) exerted significant cytotoxicity against Jurkat cells, while liposomes lacking either the PEO-lipid or DTA did not. Liposomes modified by the PEO-lipid with shorter PEO chain(n = 5 or 15) did not show any cytotoxicity, irrespective of their DTA-loading. The cytotoxicity was observed even in the presence of cytochalasin B, an inhibitor of endocytosis. Judging from these results, we concluded that the PEO-lipid(n = 32)-modified liposome directly fused with plasma membrane of Jurkat cell.

Direct extraction of A and B blood group antigens from human red cells by liposomes.

BACKGROUND: Some of the major blood group antigens are on lipids and proteins of the red cell membrane. Incubation of intact red cells with liposomes containing specially designed artificial lipids has been shown to result in the extraction of membrane proteins by the liposomes. The extraction of blood group structures and the retention of their antigenicity have not been reported. STUDY DESIGN AND METHODS: After the incubation of red cells with liposomes, the extraction of the antigens from human red cells by liposomes was examined by evaluation of the agglutination of the liposomes by respective antisera. RESULTS: Agglutination specific to the A and B blood group antigens was seen, which indicated that the antigenicity of the blood group antigens was retained even after the extraction by the liposomes. The presence of an artificial boundary lipid, 1,2-dimyristamido-1,2-deoxyphosphatidylcholine, in the liposome was crucial to the efficient extraction of the A and B antigens. On the other hand, the extraction of D, M, N, and P1 was not always detectable by agglutination. CONCLUSION: The A and B blood group antigens were directly extracted from red cells by liposomes without loss of antigenicity.

0/w-emulsion of alpha-linolenic acid stabilized with hydrophobized polysaccharide. Its effect on the growth of human colon cancer cells.

To pursue a systemic administration of alpha-linolenic acid (ALA), which is a selective cytotoxic agent, we formulated an ALA o/w-emulsion stabilized by cholesterol-bearing pullulan (CHP-55-2.1) and trioctanoylglyceride (TriC8). This emulsion was stable even in the presence of bovine serum albumin (BSA). Peroxidation of ALA was drastically depressed by the emulsification using CHP. In addition, cytotoxic effect of the CHP/ALA/TriC8-emulsion against human colon cancer cell (RPM14788) was much higher than that of free ALA. However, no significant difference was observed in cell internalization efficiency of ALA between the two. These results suggest that difference in the cytotoxicity between the CHP/ALA/TriC8-emulsion and free ALA may come from difference in the intracellular behavior of ALA.

Endocytosis of poly(ethylene oxide)-modified liposome by human lymphoblastoid cells.

Egg PC liposome as reconstituted with poly(ethylene oxide)-bearing lipid (coded as PEO-lipid (n = 15)) was remarkably endocytosed by Jurkat cell, which was a lymphoblastoma derived from human T cell. To confirm the endocytosis, two kinds of fluorescent probes (FITC-dextran and octadecyl rhodamine B) were employed. The former was loaded in the aqueous phase of the liposome, while the latter was embedded in the liposomal membrane. Both probes were found coincidentally at the same site in the cytosol, clearly suggesting that whole liposome entered the cell. The endocytosis was most obvious when PEO-lipid (n = 15) was employed above 50 mol%. FITC-Dextran entered the cell was found small dots in the cell, not dispersive. Even when octadecyl rhodamine B was used, no membrane fluorescence was observed at all. The uptake closely related to the cell metabolism as affected by the culture temperature and serum in the incubation medium. Furthermore, the addition of cytochalasin B completely prohibited the cell uptake of liposomes.

Partition of polysaccharide-coated liposomes in aqueous two-phase systems.

Hydrophobized polysaccharides such as cholesterol-bearing pullulan (CHP), dextran (CHD) and mannan (CHM) effectively coat the liposomal surface. Partition of the hydrophobized polysaccharide-coated liposomes in an aqueous two-phase system (PEO (top)/pullulan (bottom) or PEO (top)/dextran (bottom)) was investigated (PEO = poly(ethylene oxide)). Conventional liposomes without a polysaccharide coat mostly locate at the interface between the two polymer phases. The polysaccharide-coated liposomes, on the other hand, were partly partitioned to the bottom polysaccharide phase depending on the structure of the hydrophobized polysaccharide on the liposomal surface. The affinity between the polysaccharide on the liposomal surface and that in the bulk bottom phase controls the efficiency of partition. The sequence of interaction strength between the two carbohydrates was the following: for the PEO/dextran two-phase system, dextran(liposome)-dextran(bulk) > mannan(liposome)- dextran(bulk) > pullulan(liposome)-dextran(bulk); while for the PEO/pullulan system, the sequence of interaction strength was pullulan(liposome)- pullulan(bulk) > dextran(liposome)-pullulan(bulk) approximately mannan(liposome)-pullulan(bulk).

Interaction between ganglioside-containing liposome and rat T-lymphocyte: confocal fluorescence microscopic study.

Interaction between rat T-lymphocytes and a ganglioside (GM3, GD3, GT1b, or GQ1b)-containing liposome was investigated in vitro. The direct stimulation of T cell by a ganglioside-containing liposome was followed by monitoring an increase in the intracellular calcium signal using a confocal laser fluorescence microscopic method. The GT1b- or GQ1b-containing liposome strongly stimulated the cell, while the GM3- or GD3-containing liposome showed much less effect. Free gangliosides did not stimulate the cell at all under the same condition. The extent of the stimulation depended on the surface density of GT1b on the liposome accompanied by a clear threshold concentration. The efficiency of this direct T cell stimulation with the ganglioside-containing liposome was closely related to the efficiency of the tumor growth suppression reported previously by ourselves.

Transfer of membrane proteins from human platelets to liposomal fraction by interaction with liposomes containing an artificial boundary lipid.

The direct transfer of membrane proteins from human platelets to the liposomal fraction was examined, particularly in relation to platelet activation during the process. The incorporation of an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), in the interacting liposome considerably enhanced the efficiency of the protein transfer. The transfer proceeded with neither significant activation nor lysis of the platelet, and the activation of the platelet with thrombin did not affect the amount of the transferred proteins. A wide range of platelet membrane proteins was transferred, and they were almost comparable to those in a sample prepared by glycerol lysis/centrifugation. In addition, they included the major surface glycoproteins GPIIb and GPIIIa without noticeable contamination of soluble cytosol proteins. The protein transfer method is a one-pot process and clearly more convenient than the conventional 'extract and reconstitute' approach. These results strongly support the use of the transfer process, especially with DDPC, as an alternative to the conventional detergent-solubilization or the solvent-extraction methods for preparation of samples of platelet membrane proteins.

Taste receptor proteins directly extracted by liposome from intact epithelium of bullfrog tongue.

This work first provides that epithelial membrane proteins can be directly transferred from animal intact tissue to liposome. Bullfrog tongue was treated with a specially modified liposome that contains an artificial boundary lipid. Glossopharyngeal nerve responses of the treated tongue were then measured to five taste stimuli (NH4Cl, L-Ala, sucrose, L-Leu, and quinine hydrochloride). The liposomal treatment caused remarkable changes of the taste nerve responses. Gel electrophoretic analysis of the treated liposome revealed that the direct transfer of proteins, likely taste receptor, certainly occurred from the tongue epithelium to the liposome.