One-step formulation of nonionic surfactant bicelles (NSBs) by a double-tailed polyglycerol-type nonionic surfactant.

Bicelles are generally formed by phospholipid-based systems and are useful for various applications, such as nanocarriers or membrane protein crystallization. The same disc-like assemblies, nonionic surfactant bicelles (NSBs), can also be formed using nonionic amphiphiles, but this has not been reported extensively. We report a novel NSB system that employs the double-tailed nonionic amphiphile, polyglyceryl dialkyl ether (C12CmGn), which has two alkyl chains and a polyglyceryl group. A symmetric-tail molecule, C12C12G13.8, formed vesicles, whereas an asymmetric-tail molecule, C12C14G15.5, formed NSBs through a simple one-step process using ultrasonication. The 1 wt% aqueous solution of C12C14G15.5 was in a two-phase equilibrium of a lamellar phase and a water phase. Transparent dispersion was obtained through ultrasonication treatment. The size distribution in the dispersion was obtained by dynamic light scattering (DLS), resulting in a narrow distribution of around 20 nm in diameter. A negatively-stained transmission electron microscopy (TEM) image showed oblong and spherical shapes, which are typically observed in bicelle-forming systems. A small angle neutron scattering (SANS) measurement well proved bicelle formation by fitting a core-shell bicelle form factor model. The disc thickness and diameter were in agreement with the values obtained by DLS and TEM, respectively. A larger shell thickness at the rim part than at the flat disc part suggested that NSB aggregates have inhomogeneous molecular distribution. Similar to phospholipid systems, the bicelle-forming C12C14G15.5 system produced a defective lamellar phase formation at high surfactant concentrations, whereas a general lamellar phase was formed in the vesicle-forming C12C12G13.8 system.

Formation of bilayer membrane and niosomes by double-tailed polyglyceryl-type nonionic surfactant.

Vesicles with synthetic nonionic surfactants are called niosomes or NSVs, and these have been the focus of attention as an alternative to phospholipid liposomes as drug carriers. Especially it is demanded to discover novel niosomal systems with polyol-type nonionic surfactants from the viewpoint of environmental aspects. In this paper, a novel series of double-tailed nonionic surfactants, polyglyceryl dialkyl ethers, (C12)2Gn (n = 2.3, 5.4, 9.4, and 13.8), was synthesized, and its aqueous phase behavior and niosome formation were studied. Because of its double-tailed molecular structure, a lamellar liquid crystalline phase was dominant in the binary phase diagrams for different polyglyceryl chain lengths. The single lamellar liquid crystalline phase region was expanded as the polymerization degree in the hydrophilic moiety increased. Small-angle X-ray scattering spectra revealed the lamellar structure for the (C12)2G2.3 was extremely loose. Molecular packing in the lamellar phase was analyzed except for the (C12)2G2.3 system by using a geometrical model of the lamellar phase. The effective cross-sectional area per molecule at the interface increased extensively as dilution for the (C12)2G13.8 system but remained almost unchanged for the (C12)2G5.4 system. From the molecular parameters, water-holding ability in the lamellar phase was evaluated, and the results indicated strong hydration ability of the long polyglyceryl chain. In a dilute region, micron-sized giant niosomes and small niosomes of about 100 nm were formulated by vortex mixing and ultrasonication, respectively. The multilamellar structure of the small niosomes was confirmed by transmission electron microscopy. Cholesterol addition in the present surfactant lamellar phase induced the phase transition to the liquid ordered phase, which is the same phenomenon in a phospholipid-cholesterol mixture. The stability of niosomes with/without cholesterol was monitored by the niosome size change. In both cases, the niosomes were stable for at least 100 days.

Dual signal-responsive liposomes for temperature-controlled cytoplasmic delivery.

For production of a new type of functional liposome whose destabilization can be triggered by a combination of a temperature signal and acidic pH signal, we prepared liposomes modified with hyperbranched poly(glycidol) derivatives having N-isopropylamide and carboxyl groups. HeLa cells incubated with the dual signal-responsive liposomes encapsulating a water-soluble fluorescent dye pyranine at 28 °C displayed punctate fluorescence of pyranine, indicating that the liposomes were trapped in endosome. However, after heating at 45 °C for 15 min, the same cells exhibited diffuse fluorescence of pyranine, indicating that destabilization of the liposomes in endosome with an acidic environment in combination with the brief heating caused efficient transfer of the contents into cytosol. The dual signal-responsive liposomes might have usefulness for site-specific delivery of membrane-impermeable molecules, which exhibit bioactivities in the intracellular spaces, such as siRNA and proteins.

Synthesis and characterization of hyperbranched poly(glycidol) modified with pH- and temperature-sensitive groups.

Hyperbranched poly(glycidol)s with varying degrees of polymerization were modified by reaction with succinic anhydride and isopropylamine to obtain novel pH- and thermosensitive polymers. These polymers exhibited phase transitions in response to decreasing pH and/or increasing temperature, depending on the degree of polymerization and the ratio of succinyl group to N-isopropylamide. It was possible to harvest a bioactive molecule, rose bengal, from solution using the phase transition of thermosensitive hyperbranched poly(glycidol).

Highly Viscoelastic Reverse Wormlike Micellar Systems from a Mixture of Lecithin, Polyglycerol Fatty Acid Monoesters, and an Oil.

We report new lecithin reverse wormlike micelles with high viscoelasticity formed using lecithin/polyglycerol fatty acid monoester (PGLFA)/oil systems. In this study, the influence of the amphiphilicity (i.e., hydrophile-lipophile balance, HLB) of PGLFA on the phase behavior and rheological properties of reverse wormlike micelles was investigated in detail. PGLFAs with degrees of polymerization of polyglycerol varying between 6-40 and constituent fatty acids with chains between 6-18 carbon atoms long were used. Partial phase diagrams of the lecithin/PGLFA/n-decane systems indicated that the appropriate PGLFA could change the lecithin/oil solution into a highly viscoelastic solution comprising reverse wormlike micelles. Rheological measurements showed that all systems that formed reverse wormlike micelles exhibited an unusual phenomenon called "shear-thickening". Furthermore, reverse wormlike micelles grew as the PGLFA concentration increased and the zero-shear viscosity (η0) of the solution rapidly increased. Our results indicate that the magnitude of the maximum η0 depends on the degree of polymerization of the constituent polyglycerol in the PGLFA, while the size of the reverse micellar region and the highly viscous region in the phase diagram depends on the HLB value of the PGLFA.

A liposome-based antigen delivery system using pH-sensitive fusogenic polymers for cancer immunotherapy.

Highly pH-sensitive liposomes that deliver antigenic molecules into cytosol through fusion with or destabilization of endosome were prepared by surface modification of egg yolk phosphatidylcholine/dioleoylphosphatidylethanolamine (1/1, mol/mol) liposomes with 3-methylglutarylated poly(glycidol) of linear (MGlu-LPG) or hyperbranched structure (MGlu-HPG). These polymer-modified liposomes were stable at neutral pH, but they became strongly destabilized below pH 6, which corresponds to the pH of endosome. These polymer-modified liposomes were taken up by murine dendritic cells (DCs) more efficiently than the unmodified liposomes were through an endocytic pathway. They introduced entrapped ovalbumin (OVA) molecules into cytosol. Subcutaneous or nasal administration of the polymer-modified liposomes loaded with OVA induced generation of OVA-specific cytotoxic T cells (CTL) much more effectively than the unmodified liposomes loaded with OVA. Furthermore, administration of the polymer-modified OVA-loaded liposomes to mice bearing E.G7-OVA tumor significantly reduced the tumor burden, although the OVA-loaded unmodified liposomes only slightly affected tumor growth. Results suggest that the polymer-modified liposomes with highly pH-sensitive destabilizing property are promising as antigen carriers for efficient cancer immunotherapy.

New lecithin organogels from lecithin/polyglycerol/oil systems.

New liquid substances that induce the formation of lecithin organogels composed of reverse worm-like micelles were studied. The phase behavior and rheological properties of lecithin/polyglycerol (PGL)/oil systems were investigated in detail; the polymerization degrees of the glycerol residues were 3, 4, 6, 10, 20, and 40. From the partial phase diagrams of the lecithin/PGL/n-decane systems, it was apparent that highly viscoelastic reverse worm-like micelles formed upon the addition of small amounts of the PGL, except in the case of the PGL with a polymerization degree of 40. Steady-flow viscosity measurements showed that the zero-shear viscosity (η0) of the reverse worm-like micelles rapidly increased with the concentration and polymerization degree of the PGLs, reaching a maximum value that was 750,000 times the viscosity of n-decane and thus resulting in the growth of these micelles. It is noteworthy that the η0 values of lecithin organogels formed using PGLs were higher than the η0 value of the lecithin organogel formed using glycerol (GL). From dynamic viscoelasticity measurements, it was shown that the viscoelastic behavior of the reverse worm-like micelles was consistent with the single Maxwell model, which is the basic model of a viscoelastic body. It follows from this study that PGLs are useful liquids because they can induce the formation of lecithin organogels with high viscoelasticity, as do other liquids such as water, glycerol, ethylene glycol, and formamide.

Carboxylated hyperbranched poly(glycidol)s for preparation of pH-sensitive liposomes.

Previous reports by the authors described intracellular delivery using liposomes modified with various carboxylated poly(glycidol) derivatives. These linear polymer-modified liposomes exhibited a pH-dependent membrane fusion behavior in cellular acidic compartments. However, the effect of the backbone structure on membrane fusion activity remains unknown. Therefore, this study specifically investigated the backbone structure to obtain pH-sensitive polymers with much higher fusogenic activity and to reveal the effect of the polymer backbone structure on the interaction with the membrane. Hyperbranched poly(glycidol) (HPG) derivatives were prepared as a new type of pH-sensitive polymer and used for the modification of liposomes. The resultant HPG derivatives exhibited high hydrophobicity and intensive interaction with the membrane concomitantly with the increasing degree of polymerization (DP). Furthermore, HPG derivatives showed a stronger interaction with the membrane than the linear polymers show. Liposomes modified with HPG derivatives of high DP delivered contents into the cytosol of DC2.4 cells, a dendritic cell line, more effectively than the linear polymer-modified liposomes do. Results show that the backbone structure of pH-sensitive polymers affected their pH-sensitivity and interaction with liposomal and cellular membranes.

Cycloheptyne intermediate in the reaction of chiral cyclohexylidenemethyliodonium salt with sulfonates.

Reactions of (R)-4-methylcyclohexylidenemethyl(phenyl)iodonium salt and its 3-trifluoromethylphenyl and 4-methoxyphenyl derivatives (1) with tetrabutylammonium mesylate and triflate were carried out in chloroform at 60 degrees C. The products include (S)-4-methylcyclohexylidenemethyl sulfonate (2) and (R)-5-methylcyclohept-1-enyl sulfonate (3) as well as iodoarene. Reactions of (S)-1 were confirmed to provide the counterpart results. The rearranged triflate (R)-3Tf formed in the reaction with triflate maintains mostly the ee (enantiomeric excess) of (R)-1, while the ee of the mesylate product 3Ms is largely lost. The (13)C-labeling at the exocyclic position of 1 results in the isotopic scrambling of C-1 and C-2 of 3Ms in the mesylate reaction. The degree of the scrambling agrees well with that of the loss of ee of (R)-3Ms obtained from (R)-1, implying that the racemization is not due to the intermediate formation of achiral, primary 4-methylcyclohexylidenemethyl cation. Reaction of 1 with mesylate in the presence of CH(3)OD provided the 3Ms deuterated at the 2-position. When tetraphenylcyclopentadienone was added to the mesylate reaction system, the adduct of the 4-methylcycloheptyne intermediate was obtained in 24% yield, but the normal products 2Ms and 3Ms were still formed. The 3Ms obtained here in a low yield maintains the high ee of 1. These results indicate that the cycloheptyne is an intermediate responsible for the formation of racemic product 3Ms in the mesylate reaction. It is also concluded that the unrearranged products 2 are formed via the competitive pathways of in-plane and out-of-plane S(N)2 reactions.

Solvolysis of 4-methylcyclohexylidenemethyliodonium salt: chirality probe approach to a primary vinyl cation intermediate.

Optically active 4-methylcyclohexylidenemethyl(aryl)iodonium tetrafluoroborate (1.BF(4)(-)) was prepared and its solvolysis was carried out at 60 degrees C in various solvents. The main product is optically active 4-methylcycloheptanone (or its enol derivative) in unbuffered solvents, accompanied by the iodoarene. The rearranged product always maintains the optical purity of the starting 1. Its stereochemistry conforms to a mechanism involving the rearrangement via the sigma-bond participation in departure of the nucleofuge, followed by trapping of the resulting chiral 5-methylcyclohept-1-enyl cation with a nucleophilic solvent. That is, the achiral, primary vinyl cation is not involved during the reaction. The unrearranged substitution product is also obtained in a minor fraction in unbuffered methanol, ethanol, and acetic acid, but not in trifluoroethanol or hexafluoro-2-propanol: the methoxy product from methanolysis is largely racemized, but the acetolysis product is obtained mainly via retention of configuration. Reactions of 1 with bromide, acetate, and trifluoroacetate in chloroform give unrearranged substitution products in different degrees of inversion. These unrearranged products are concluded to be formed via the direct nucleophilic substitutions. Added bases such as sodium acetate in methanol lead to the unrearranged methoxy products of complete racemization, which is ascribed to the alpha elimination (to give an alkylidenecarbene) followed by the solvent insertion.

Elimination-addition mechanism for nucleophilic substitution reaction of cyclohexenyl iodonium salts and regioselectivity of nucleophilic addition to the cyclohexyne intermediate.

The reaction of 4-substituted cyclohex-1-enyl(phenyl)iodonium tetrafluoroborate with tetrabutylammonium acetate gives both the ipso and cine acetate-substitution products in aprotic solvents. The isomeric 5-substituted iodonium salt also gives the same mixture of the isomeric acetate products. The reaction is best explained by an elimination-addition mechanism with 4-substituted cyclohexyne as a common intermediate. The cyclohexyne formation was confirmed by deuterium labeling and trapping to lead to [4 + 2] cycloadducts and a platinum-cyclohexyne complex. Cyclohexyne can also be generated in the presence of some other mild bases such as fluoride ion, alkoxides, and amines, though amines are less effective bases for the elimination. Kinetic deuterium isotope effects show that the anionic bases induce the E2 elimination (k(H)/k(D) > 2), while the amines allow formation of a cyclohexenyl cation in chloroform to lead to E1 as well as S(N)1 reactions (k(H)/k(D) approximately 1). Bases are much less effective in methanol, and methoxide was the only base to efficiently afford the cyclohexyne intermediate. Nucleophiles react with the cyclohexyne to give regioisomeric products in the ratio dependent on the ring substituent. The observed regioselectivity of nucleophilic addition to substituted cyclohexynes is rationalized from calculated LUMO populations, which are governed by the bond angles at the acetylenic carbons: The less deformed carbon has a higher LUMO population and is preferentially attacked by the nucleophile.