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.

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.

Liposomal membrane. I. Chemical damage of liposomal membranes with functional detergent.

The interaction and reaction between liposomal membrane and a functional detergent, N-hexadecyl-N-(imidazol-4-yl)methyl-N,N-dimethylammonium chloride hydroperchlorate (Im-I), have been investigated in conjunction with the leakage of bromothymol blue encapsulated as a marker in the bilayers of liposomes. Im-I carries an imidazole moiety and was expected to behave as a simple lipase model. The reaction with Im-I significantly enhanced the leakage of bromothymol blue encapsulated in the egg lecithin and dipalmitoyl phosphatidylcholine liposomes. During the course of reaction with Im-I, the formation of acyl-imidazole intermediate was clearly identified, which was certainly connected with the bromothymol blue release. From various kinetic results on bromothymol blue release and acyl-imidazole formation, it has been suggested that the bromothymol blue release from liposomal bilayer may be caused by the local and instantaneous decomposition of lipids when Im-I penetrates into the bilayer. However, it has also been demonstrated that the immediate reconstruction of liposomes retains the barrier function to protect against the further release of bromothymol blue.

Deuterium nuclear magnetic resonance studies on the interaction of glycophorin with 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine.

Membrane dynamics of dimyristoylphosphatidylcholine (DMPC) lipid bilayer which contains glycophorin with an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), was investigated by 2H-NMR technique. For this purpose, both DMPC and DDPC were deuterated at the position of the 8th carbon atom of their acyl chains. Comparing, with DMPC bilayers, DDPC bilayers showed larger deuterium quadrupole splitting (delta nu rho) by approx. 2 kHz. This was explicable in terms of the stabilization of the membrane due to the formation of a strong hydrogen bonding in bilayers. Addition of glycophorin to the DMPC or DDPC single bilayers caused an increase in the delta nu rho value. The delta nu rho value of DMPC/DDPC mixed lipid bilayer was smaller than that of each single lipid bilayer DDPC in the DDPC/DMPC mixed bilayer was not phase-separated but homogeneously distributed. In glycophorin-reconstituted DMPC-d4/DDPC mixed bilayers, the delta nu rho of DMPC-d4 was almost identical to that of the simple DMPC-d4 bilayer. On the other hand, the delta nu rho of DDPC-d4 in the DMPC/DDPC-d4 mixed bilayer increased significantly upon the reconstitution of glycophorin. Judging from these data, we concluded that, in the DDPC/DMPC mixed bilayer which contains glycophorin, DMPC simply behaves as the matrix lipid, while DDPC surrounds glycophorin and certainly plays a role of the boundary lipid.

Activity of bile-salt-stimulated human milk lipase in the presence of liposomes and mixed taurocholate-phosphatidylcholine micelles.

(1) The interaction of bile-salt-stimulated human milk lipase and liposomal membranes has been investigated in the presence or absence of sodium taurocholate. Freshly purified enzyme enhances the permeability of liposomal membranes but thermally inactivated enzyme does not. (2) The ability of the enzyme to catalyze the hydrolysis of a relatively hydrophilic substrate, 4-nitrophenyl acetate, and a more hydrophobic substrate, 4-nitrophenyl palmitate, has also been measured in media containing small unilamellar vesicles of egg phosphatidylcholine in both the absence and presence of taurocholate, and also in the presence of free taurocholate in the absence of liposomes. (3) The enzyme-catalyzed hydrolysis of 4-nitrophenyl acetate is enhanced in all of these systems, but 4-nitrophenyl palmitate is protected from enzymic attack in the phosphatidylcholine-bile salt systems. If free taurocholate be present in the system before 4-nitrophenyl palmitate is added, then, and only then, is enzymic activity observed. (4) These results have been interpreted in terms of the importance of the microenvironment around the substrate and the role played by the bile salt surfactant in stimulating the enzyme.

The mechanism of liposomal damage by taurocholate.

The stability of small unilamellar vesicles formed by egg-yolk phosphatidylcholine (PC) has been examined in the presence of sodium taurocholate. The permeability of the vesicular membrane changes as the total taurocholate concentration increases, until a transformation from mixed bile salt/PC vesicles to mixed micelles occurs. Based on experiments in which the bile salt-induced release of either hydrophilic (carboxyfluorescein) or hydrophobic (Bromothymol blue) probes was studied, and on fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene and turbidity measurements, a two-step process for the initial stage of liposomal damage by taurocholate is postulated.

Liposomal membranes. XX. Autoxidation of unsaturated fatty acids in liposomal membranes.

Autoxidation of egg phosphatidylcholines and polyunsaturated fatty acids, arachidonic and linoleic acids, has been investigated in homogeneous and liposomal membrane systems. In order to monitor the very initial stage of the radical chain mechanism of the autoxidation, an improved method using 1,1-diphenyl-2-picrylhydrazyl radical was newly implemented and used with the regular thiobarbituric acid test to determine the peroxides of polyunsaturated fatty acids. Autoxidation of polyunsaturated fatty acids or lipids was significantly enhanced in liposomal bilayers compared to that in a bulk homogeneous solution. In liposomal bilayers, the reaction could be controlled by membrane fluidity, which was confirmed by the fluorescence polarization technique using 1,6-diphenylhexatriene and dansylhexadecylamine. Even vitamin E esters such as the acetate and the pivalate could depress effectively the autoxidation of egg phosphatidylcholines in bilayer systems, which supported Lucy's proposal (Diplock, A.T. and Lucy, J.A. (1973) FEBS Lett. 29, 205-210) about the importance of the side-chain of vitamin E as an antioxidant.

Synthesis and characterization of 1,2-dimyristoylamido-1, 2-deoxyphosphatidylcholine as an artificial boundary lipid.

The synthesis and characterization of an artificial boundary lipid, 1,2-dimyristoylamido-1,2-deoxyphosphatidylcholine (DDPC), are described. DDPC has two amide bonds instead of ester bonds of regular lecithins such as 1,2-dimyristoylphosphatidylcholine (DMPC). In differential scanning calorimetry (DSC) measurements, DDPC gave two endothermic peaks: one was at 18.0 degrees C (delta H = 10.74 kJ.mol-1) and the other at 23.0 degrees C (delta H = 12.91 kJ.mol-1). The former peak was sharp and considered to be the phase transition of the hydrocarbon region, while the latter was assigned to the melt of the hydrogen-belt formed by the amide groups of DDPC. Addition of DDPC to DMPC made the DMPC membrane less fluid in the region close to the surface, and significantly increased the reconstitution efficiency of glycophorin into the membrane. This effect of DDPC was much larger than that of naturally occurring lipid, sphingomyelin.

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.

A newly developed immunoliposome--an egg phosphatidylcholine liposome coated with pullulan bearing both a cholesterol moiety and an IgMs fragment.

An improved methodology for providing a more stable and targetable drug carrier has been developed. This method involves the synthesis of a newly designed immunoliposome by coating the outermost surface of large oligolamellar vesicles of egg phosphatidylcholine with the polysaccharide pullulan, modified to carry both cholesterol, as the hydrophobic anchor, and the monoclonal antibody fragment (anti-sialosyl Lewis X, IgMs) as the sensory device. Compared with the binding of pullulan-coated liposomes, that of this immunoliposome to specific cells in vitro was significantly increased by factors of 447 to PC-9 and 295 to KATO-III, but only by a factor of 148 to the less specific cell, 3LL. This strong and specific binding of the immunoliposome to the cell surface of PC-9 was also confirmed by a fluorescence-microscopic investigation using the immunoliposome, which bore the hydrophobic fluorescent probe, terbium trisacetylacetonate, in the liposomal membrane.

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.

Liposomal membranes. XI. A suggestion to structural characteristics of acido-thermophilic bacterial membranes.

To understand the role of omega-cyclohexyl fatty acid residue of lipids in acido-thermophilic bacterial membranes, three unusual phosphatidylcholines, 1, 2-di-11-cyclohexylundecanoyl-L-alpha-phosphatidylcholine (11CYPC), 1,2-di-13-cyclohexyltridecanoyl-L-alpha-phosphatidylcholine (13CYPc), and 1-13-cyclohexyltridecanoyl-2-11-cyclohexylundecanoyl-L-alpha- phosphatidylcholine (1-13CY-2-11CYPC) were prepared and the steady-state fluorescence anisotropy of 1, 6-diphenylhexatriene (DPH) in the hydrophobic domain of these liposomal bilayers was determined. Compared with the case of dipalmitoyl (DPPC) or dimyristoyl phosphatidylcholine (DMPC), introducing the omega-cyclohexyl moiety onto lecithins makes the bilayers fluid below the phase transition temperature, while immobilizes them above the phase transition temperatures. The properties of the unusual phosphatidylcholine liposomes suggested by the steady-state fluorescence anisotropy investigation were in good agreement with those obtained from the thermotropic and permeability investigations. Results obtained are discussed from the view point of the role and function of lipid membranes of acido-thermophilic bacteria which contain unusual fatty acids.

Increased lung uptake of liposomes coated with polysaccharides.

Liposomes labeled with [14C]coenzyme Q10 in the lipid bilayer were coated with various polysaccharide derivatives, i.e., palmitoyl conjugates of pullulan, pullulan phosphate, amylopectin, amylopectin phosphate and amylopectin sulfate. The kinetics of disposition and the tissue distribution of [14C]coenzyme Q10 after intravenous injection of the liposomes into guinea pigs were investigated. Lung uptake of radioactivity after injection of the O-palmitoyl amylopectin- and O-palmitoyl amylopectin phosphate-coated liposomes was 5- and 3-times higher, respectively, at 30 min after injection than that of the conventional liposomes. For doubly labeled liposomes with [3H]inulin and [14C]coenzyme Q10, the 3H/14C ratios in the lung, spleen and heart were similar to one another. Urinary excretion of [3H]inulin encapsulated in O-palmitoyl amylopectin-coated liposomes was much lower than that of unencapsulated [3H]inulin. These observations suggest that the O-palmitoyl amylopectin-coated liposomes are rather stable in vivo and are taken up into tissues in the intact form.

Bile salt damage of egg phosphatidylcholine liposomes.

Physiochemical damage of egg phosphatidylcholine liposomes, caused by the salts of three bile acids, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid, has been investigated. Of the three bile salts, that of chenodeoxycholic acid was the most destructive, and the effect of the damage was examined by monitoring the induced 6-carboxyfluorescein release from the liposomes. For all three of the bile salts and under the experimental conditions, the minimum (effective) concentrations causing the 6-carboxyfluorescein release were below their critical micelle concentrations. In the case of the salt of chenodeoxycholic acid, the presence of cholesterol in the liposomal bilayers did not show any significant effect on the induced 6-carboxyfluorescein release, while, for the salts of ursodeoxycholic acid and cholic acid, the presence of cholesterol tended to depress the release. Permeation of bile salts into the membranes of liposomal bilayers made these membranes more fluid, and this fluidity was monitored by measuring the change in fluorescence polarization using 1,6-diphenylhexatriene entrapped in the liposomes. Coating the liposomes with polysaccharides, to make them more hydrophobic, led to their easier lysis by the bile salts.

Configuration of the active Mg-ATP complex in protein kinase C reaction.

To probe the active site structure of protein kinase C stereochemical studies were carried out by using ATP beta S. The enzyme utilizes either one of the diastereomers (SP and RP) of ATP beta S almost equally well as a substrate. This result contrasts with that for cyclic AMP-dependent protein kinase, suggesting that the topography of the nucleotide-binding site is significantly different between the two kinases.

Protein-coated and polysaccharide-coated liposomes as drug carriers.

Saccharides on the surface of cell membranes play an important role in cell-cell recognition, which is the most important process utilizable for targeting of drugs encapsulated in an artificial cell, liposome. To design a targetable drug carrier, hence, employing synthesized or natural glycolipids as the recognition site of the liposomal drug carrier is certainly one of useful approach. On the other hand, coating the outermost surface of liposomes with polysaccharide derivatives is also another way for liposomes to be utilized as a targetable drug carrier. In this review, from such a viewpoint, the importance and usefulness of saccharide moiety on the surface of liposomes will be discussed in conjunction with the targeting of drugs.

Interaction of flavonoids with 1,1-diphenyl-2-picrylhydrazyl free radical, liposomal membranes and soybean lipoxygenase-1.

The interaction of the antiperoxidative flavonoids namely, quercetin, quercetrin, rutin, myricetin, phloretin, phloridzin, catechin, morin and taxifolin with the 1,1,-diphenyl-2-picrylhydrazyl (DPPH) free radical was demonstrated. Flavonoid-DPPH interaction was looked at in the absence and presence of liposomes so as to reveal some information on bilayers. Perturbations in the lipid bilayers were monitored with the fluorescent probe, dansylhexadecylamine (DSHA). It was observed that the interaction of the flavonoids on the lipid bilayer occurred in the polar zone of the lipid bilayers. The flavonoids were able to scavenge free radicals and could do so in biomembranes. It is suggested that the DPPH free radical abstracts the phenolic hydrogen of the flavonoid molecule and that this could be the general mechanism of the scavenging action of the antiperoxidative flavonoids. The effects of the flavonoids on soybean lipoxygenase-1 were investigated both in buffer and also in liposomal suspension. All the flavonoids studied showed inhibition of the enzyme in both systems but the inhibition was greater in the liposomal suspension. Quercetin was the most potent and it inhibited the lipoxygenase in the liposomal suspension by about 42% while the other flavonoids inhibited the enzyme by about 14-23%. We observed that the effect of myricetin and quercetin on the enzyme was pH dependent.

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.

Liposomal membranes. XII. Adsorption of polysaccharides on liposomal membranes as monitored by fluorescence depolarization.

Under specific conditions where neither induced aggregation nor fusion is brought about, the adsorption of polysaccharides on liposomal membranes was investigated by the fluorescence depolarization technique using fluoresceinylthiocarbamoyl-dextrans (FITC-dex) as probes. The adsorption of FITC-dex on liposomes significantly increased the fluorescence polarization of FITC fluorophore due to the restriction of the mobility of the dextrans. Dextran with larger molecular weight was efficiently adsorbed on liposomes, in good agreement with the tendency for polysaccharide-induced aggregation of liposomes (Sunamoto et al. (1980) J. Biochem. 88, 1219--1226). The adsorption seemed to be related to the fluidity of liposomal membranes, since dextrans were adsorbed more efficiently on egg lecithin liposomes than dipalmitoyl lecithin liposomes at 25.0 degrees C. However, the adsorption of dextrans did not cause a significant change in the fluidity of the liposomal membrane itself under the specific conditions adopted in this work; this was ascertained from the mobility of sodium 8-anilino-1-naphthalenesulfonate (ANS) intercalated close to the surface of liposomes.

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.