Junzo Sunamoto

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.

Supramolecular assembly of hydrophobized polysaccharides

Abstract Hydrophobized polysaccharides, which were partly substituted by hydrophobes such as cholesterol, formed a hydrogel nanoparticle by their self-assembly in water. The size, density and colloidal stability of the nanoparticle were controlled by changing the substitution degree of hydrophobes and the hydrophobicity. The hydrophobized polysaccharides were further functionalized by conjugation of cell-specific saccharide, thermoresponsive polymer or polyethylene oxide. The hydrophobized polysaccharides interacted with various soluble proteins or other molecular assemblies, such as monolayer, black lipid membrane, liposome and oil-in-water emulsion. Such supramolecular assemblies with hydrophobized polysaccharides were utilized in biotechnology and medicine.

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.

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.

HER2-Specific T-Cell Immune Responses in Patients Vaccinated with Truncated HER2 Protein Complexed with Nanogels of Cholesteryl Pullulan

Purpose: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4+ T cells in HLA-A2402-positive patients with therapy-refractory HER2-expressing cancers. Experimental Design: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 μg of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4+ T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein. Results: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade 1 transient skin reaction at the sites of vaccination. HER2-specific CD8+ and/or CD4+ T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom both T-cell responses were detected in these patients. In four patients with CD8+ T-cell responses, two patients reacted to previously identified HER263-71 peptide and the other two reacted only to 146HER2 mRNA-transduced cells. Conclusions: CHP-HER2 vaccine was safe and induced HER2-specific CD8+ and/or CD4+ T-cell immune responses.

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.

Liposomal membranes. XV: Importance of surface structure in liposomal membranes of glyceroglycolipids

Abstract To elucidate the importance of the headgroup structure of glycolipids in the physicochemical properties of liposomal membranes of glycolipids, two diglucosyldialkylglycerols containing an α(1′ → 4′) or a β(1′ → 4′) glucoside linkage, 1,2- dihexadecyl -O-β- d -maltosyl (1′ → 3)-rac- glycerol (MAL-DG) and 1,2- dihexadecyl -O-β- d -cellobiosyl (1′ → 3)-rac- glycerol (CEL-DG) were employed. The fluorescence spectra and steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in the deep hydrophobic domain of these liposomal bilayers and dansylhexadecylamine in the vicinity of glycerol backbone were measured, respectively. Compared with dipalmitoylphosphatidylcholine (DPPC) liposomes, the phase-transition temperatures (Tc) of the present two glycolipid liposomes were about 11–15°C higher and the fluidity at the surface of these glycolipid liposome was considerably lower. This means that the interaction between neighboring diglucoside headgroups may be stronger than that between phosphatidylcholine headgroups. Fluorospectrometric and CPK model studies suggested that the structural difference in anomerization and epimerization of the disaccharide moiety of glyceroglycolipids is an important factor for determining the physicochemical properties of these glycolipid liposomes.

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

Abstract 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.

Improved stability of black lipid membranes by coating with polysaccharide derivatives bearing hydrophobic anchor groups

Abstract Black lipid membranes were coated with modified polysaccharides bearing hydrophobic palmitoyl and cholesteryl moieties. The changes in membrane structure were investigated using dipicrylamine, a lipophilic ion, as membrane probe. The kinetics of ion transport through the black lipid membranes were studied using the charge pulse relaxation technique. With this technique it was found that it is possible to detect the insertion of the hydrophobic anchor groups of the polysaccharides into the membrane bilayer. As a result of the surface coating, these membranes exhibit a drastically increased long-term stability.

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.