Keita Hayashi

Systematical Characterization of Phase Behaviors and Membrane Properties of Fatty Acid/Didecyldimethylammonium Bromide Vesicles

Fatty acids (FAs) are known to form vesicle structures, depending on the surrounding pH conditions. In this study, we prepared vesicles by mixing FAs and a cationic surfactant, and then investigated their physicochemical properties using fluorescence spectroscopy and dielectric dispersion analysis (DDA). The assemblies formed from oleic acid (OA) and linoleic acid (LA) were modified by adding didecyldimethylammonium bromide (DDAB). The phase state of FA/DDAB mixtures was investigated with pH titration curves and turbidity measurements. The trigonal diagram of FA/ionized FA/DDAB was successfully drawn to understand the phase behaviors of FA/DDAB systems. The analysis of fluidities in the interior of the membrane with use of 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated that the membrane fluidities of OA/DDAB and LA/DDAB at pH 8.5 slightly decreased in proportion to the molar ratio of DDAB in FA/DDAB systems. The fluorescent probe 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated that the LA vesicle possessed a dehydrated surface, while the OA vesicle surface was hydrated. Modification of LA vesicles with DDAB induced the hydration of membrane surfaces, whereas modification of OA vesicles by DDAB had the opposite effect. DDA analysis indicated that the membrane surfaces were hydrated in the presence of DDAB, suggesting that the surface properties of FA vesicles are tunable by DDAB modification.

Enhanced cytotoxicity for colon 26 cells using doxorubicin-loaded sorbitan monooleate (Span 80) vesicles.

Span 80 (sorbitan monooleate) vesicles behaved differently from conventional phospholipid vesicles (liposomes) because the former had a more fluid interface. After doxorubicin hydrochloride (DOX) was encapsulated into the Span 80 vesicle (loading efficiency: 63 %), DOX-loaded Span 80 vesicles (DVs) were thereafter added to Colon 26 cells. It was suggested, from the flow cytometric analysis and confocal laser microscopic observation, that DVs directly deliver DOX into the cytoplasm of Colon 26 cells. DVs showed the different delivery manner from the DOX-loaded liposomes (DLs). It is considered that the difference of delivery manner between DVs and DLs resulted in the difference of cytotoxicity (IC50); i.e. IC50 values for DVs and DLs were 5 and > 30 μM, respectively. The results obtained herein would give the fundamental findings which can contribute to the improvement of formulation of conventional liposome-based carrier and its cytotoxicity.

Relationship between the mobility of phosphocholine headgroup and the protein-liposome interaction: a dielectric spectroscopic study.

Proteins could affect the headgroup mobility of phospholipid within liposome membranes through the protein-liposome interaction. The variation of headgroup mobility of phospholipid was then investigated by using the dielectric dispersion analysis. The eight proteins (Mw = 4.2-28.7 kDa) were used to investigate the protein-liposome interaction. It has been revealed that the strength of the protein-liposome interaction at 25 °C was linearly correlated with the stability of intramolecular hydrogen bondings of proteins, better than with their hydrophobicity and the surface charge density. Overall, liposomes composed of binary lipid system, appeared to strongly interact with proteins, in contrast to liposomes composed of single, ternary, and quaternary lipid systems. This is probably because liposomes composed of binary lipid system favored to form the microscopic environment where proteins could interact. The present result suggested the heterogeneous phase state of lipid membranes was one of dominant factors for the interaction between proteins and lipid membranes.

Active Targeting to Osteosarcoma Cells and Apoptotic Cell Death Induction by the Novel Lectin Eucheuma serra Agglutinin Isolated from a Marine Red Alga

Previously, we demonstrated that the novel lectin Eucheuma serra agglutinin from a marine red alga (ESA) induces apoptotic cell death in carcinoma. We now find that ESA induces apoptosis also in the case of sarcoma cells. First, propidium iodide assays with OST cells and LM8 cells showed a decrease in cell viability after addition of ESA. With 50 μg/ml ESA, the viabilities after 24 hours decreased to 54.7 ± 11.4% in the case of OST cells and to 41.7 ± 12.3% for LM8 cells. Second, using fluorescently labeled ESA and flow cytometric and fluorescence microscopic measurements, it could be shown that ESA does not bind to cells that were treated with glycosidases, indicating importance of the carbohydrate chains on the surface of the cells for efficient ESA-cell interactions. Third, Span 80 vesicles with surface-bound ESA as active targeting ligand were shown to display sarcoma cell binding activity, leading to apoptosis and complete OST cell death after 48 hours at 2 μg/ml ESA. The findings indicate that Span 80 vesicles with surface-bound ESA are a potentially useful drug delivery system not only for the treatment of carcinoma but also for the treatment of osteosarcoma.

Formation of lens-like vesicles induced via microphase separations on a sorbitan monoester membrane with different headgroups.

The microphase separation of lipid molecules on a vesicle membrane can be induced, depending on the difference in the geometric structures of their headgroups. Through cryo-transmission-electron-microscopy analysis, a lens-like vesicle was prepared by mixing 50 wt% Span 40 (sorbitan monopalmitate) and 50 wt% Tween 40 [polyoxyethylene (20) sorbitan monopalmitate]. Considering the molecular structures of Span 40 and Tween 40, the high-curvature region was mainly formed by Tween 40. As determined by Fourier-transform infrared spectroscopy, dielectric-dispersion analysis, and differential scanning calorimetry, a hydration layer was likely formed because polyoxyethylene conjugates with the headgroups of Tween 40. These investigations of the obtained self-assembled aggregates of nonionic surfactants with heterogeneous surfaces could contribute to the development of new types of biomaterials.

Multi-Level Characterization of the Membrane Properties of Resveratrol-Incorporated Liposomes

Resveratrol (RES) is a type of polyphenolic compound discovered from grapes and has gained prominence as a possible contributor to many disease treatments. Herein, three different types of liposomes were prepared as model cell membranes, and then the influence of the incorporation of RES on their membrane properties was evaluated by utilizing membrane-binding fluorescent probes. The binding of RES lead to the membrane polarities decreasing slightly, regardless of the phase states of the membrane, while the membrane fluidities decreased only in the case of liquid-disordered phase. In each model membrane system, the incorporation of RES dramatically dehydrated the membrane surface, which could prevent the permeation of water-soluble materials. Fluorescence quenching of Laurdan indicated less accessibility of hydroxyl radial into the inner region of the RES-incorporated membrane. The comparison between the mass spectra of oxidized DOPC molecules treated with hydroxyl radical revealed that the RES-incorporation into DOPC membranes can contribute to prevent lipid oxidation. It is concluded that the binding of RES to the lipid membrane can play a key role in affecting membrane properties and functions.

Investigation of Fatty Acid Ketohydrazone Modified Liposome's Properties as a Drug Carrier

pH-responsive liposomes were prepared by modifying the liposome with acid-cleaving amphiphiles. Palmitic ketohydrazone (P-KH) or stearic ketohydrazone (S-KH), composed of hydrophilic sugar headgroup and hydrophobic acyl chain, was used as a modifier of the DMPC liposome. Because the ketohydrazone group of P-KH or S-KH was cleaved at low pH conditions (<pH 5.0), the delivery of the P-KH modified liposomes was observed probably via an endocytic pathway. The membrane properties of these liposomes were characterized, focusing on the variation of both polarity (measured by Laurdan) and membrane fluidity (measured by DPH) at low pH condition. The interface of the P-KH modified liposome at acidic pH was found to become more hydrophobic and less fluidic as compared with that at neutral pH; that is, P-KH modified liposome became more rigid structure. Therefore, it seems that the P-KH modified liposome could protect encapsulated drugs from the enzymes in the lysosome. This study shows the novel approach about design of pH-responsive liposomes based on the membrane properties.

Morphological Estimation of DMPC/DHPC Self-Assemblies in Diluted Condition: Based on Physicochemical Membrane Properties

Self-assembly membranes, composed of 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), were characterized at the total lipid concentration below 20 mM. The sizes of the assemblies varied depending on the molar ratio of DMPC and DHPC (q = [DMPC]/[DHPC]). The small assemblies with diameter of ca. 10 nm were formed at q ≤ 2.0 at 20 oC (below phase transition temperature of DMPC). The physicochemical membrane properties were then studied using fluorescence probes, 1,6-diphenyl-1,3,5hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, upon the dilution. DHPC micelle showed a higher membrane fluidity, while the DMPC/DHPC membranes at q ≥ 0.5 showed lower membrane fluidities as well as DMPC vesicle in gel (ordered) phase. Upon dilution, the ordered membrane properties were maintained while the solution turbidities increased, implying the morphological change of the self-assembly, bicelle to the vesicle in gel phase. Based on the obtained results, a phase diagram of DMPC/DHPC binary system (at 20 oC) is described: (i) the bicelle suspension is transparent and the membrane is in ordered state, (ii) the micelle suspension is transparent and the membrane is in disordered state, (iii) the vesicle suspension is turbid and the membrane is in ordered state. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 13 September 2018 doi:10.20944/preprints201809.0242.v1

Design of Pyrene–Fatty Acid Conjugates for Real-Time Monitoring of Drug Delivery and Controllability of Drug Release

Fluorescence probes are usually employed to analyze pharmacokinetics of drug carriers; however, this method using usual probes is not suitable to monitor drug carriers in detail because fluorescence spectra do not change by the disruption of drug carriers. In this study, pyrene–fatty acid conjugates were investigated as probes to monitor the state of drug carriers in real time. 1-Pyrenemethanol was conjugated with fatty acids, such as lauric acid, stearic acid, and behenic acid, and the conjugates were stirred in ethanol, resulting in the formation of submicron particles; these particles exhibited excimer emission. When J774.1 and Colon 26 cells were treated with these particles, the associated fluorescence spectra shifted from excimer emission to monomer emission. Moreover, the degree of change was controlled by the type of fatty acid. These results support the design of drug carriers that can be used to monitor pharmacokinetics in real time and to control the disruption time.

Characterization of the physicochemical properties of phospholipid vesicles prepared in CO2/water systems at high pressure

Phospholipid vesicles were prepared by the nonsolvent method using high-pressure CO2/water systems. The membrane properties of vesicles prepared at different pressures and temperatures were mainly characterized based on analysis of the membrane fluidity and membrane polarity, using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, respectively. The CO2(liquid)/water(liquid) and the CO2(supercritical)/water(liquid) two-phase (heterogeneous) systems resulted in the formation of vesicles with high yield (ca. 85%-88%). The membrane fluidity and polarity of the vesicles were similar to those of liposomes prepared by the conventional method. It is suggested that high-pressure CO2 can be used to form an appropriate hydrophobic-hydrophilic interface where phospholipid molecules as a self-assembled membrane.