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Photosynthetic light-harvesting polypeptide/pigment complexes (LH ) play an essential role in the primary process of an efficient solar energy-transduction in photosynthetic membrane. In our research, we aim to use the LH complex and control its direction and orientation on electrodes for the development of nanobiodevices from solar to fuel. Specifically, we focus on the construction of an array of the LH on electrodes using a modified photosynthetic protein complex prepared from modern biosynthetic manufacturing methods and in lipid membranes.

For the purpose of systemic delivery of siRNA, we previously developed polycation liposomes (PCLs) containing dicetylphosphate-tetraethylenepentamine (DCP-TEPA) as an effective siRNA carrier. In the present study, to endow these PCLs (TEPA-PCL) actively target cancer cells and angiogenic vessels, we decorated the PCLs with cyclic RGD, by using cyclic RGD-grafted distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and investigated the usefulness of this type of carrier (RGD-PEG-PCL) for active targeting. Firstly, the gene-silencing efficacy of siRNA for luciferase (siLuc2) formulated in RGD-PEG-PCL (RGD-PEG-PCL/siLuc2) was examined in vitro by using B16F10-luc2 murine melanoma cells stably expressing the luciferase 2 gene, where the siRNA was grafted with cholesterol at the 3'-end of the sense strand (siRNA-C) for the stable association of the siRNA with the PCL. RGD-PEG-PCL/siLuc2 showed high knockdown efficiency compared with siLuc2 formulated in PEGylated TEPA-PCL without cyclic RGD (PEG-PCL). Next, the gene-silencing efficacy of RGD-PEG-PCL/siLuc2 was examined in vivo by use of B16F10-luc2 lung metastatic model mice. The intravenous injection of RGD-PEG-PCL/siLuc2 showed high knockdown efficiency against metastatic B16F10-luc2 tumors in the lungs of the mice, as assessed with an in vivo imaging system. These data strongly suggest that systemic and active targeting siRNA delivery using RGD-PEG-PCL is useful for cancer RNAi therapy.

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy.

A light-harvesting (LH) antenna complex II, LHCII, isolated from spinach was immobilized onto an indium tin oxide (ITO) electrode with dot patterning of 3-aminopropyltriethoxysilane (APS) by utilizing electrostatic interactions between the cationic surface of the electrode and the anionic surface of stromal side of the LHCII polypeptide. Interestingly, the illumination of LHCII assembled onto the ITO electrode produced a photocurrent response that depends on the wavelength of the excitation light. Further, LHCII was immobilized onto a TiO2 nanostructured film to extend for the development of a dye-sensitized biosolar cell system. The photocurrent measured in the iodide/tri-iodide redox system of an ionic liquid based electrolyte on the TiO2 system showed remarkable enhancement of the conversion efficiency, as compared to that on the ITO electrode.

The two-dimensional molecular assembly was accomplished of bacteriochlorophyll a (BChl a) and zinc-substituted BChl a (Zn-BChl a) together with synthetic poly(ethylene glycol)(PEG)-linked light-harvesting (LH) model polypeptides on a gold Au(111) electrode modified with supported lipid bilayers. Model polypeptides for LH1-α from Rhodospirillum (Rs.) rubrum were successfully synthesized and stably assembled with Zn-BChl a in 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1′-glycerol)] (DOPG) lipid bilayers on an electrode at room temperature, as well as in liposomal solution, in which the Zn-BChl a complex unlike BChl a, was stably assembled. The PEG moiety of the model polypeptide assisted the stable assembly with an α-helical conformation of the LH1-α model peptides together with these pigments onto the gold electrode with defined orientation. The photocurrent response depended on the combinations of the pigments and synthetic LH model polypeptides. The results presented herein will be useful for the self-assem...

Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.

Two types of photosynthetic membrane proteins, the peripheral antenna complex (LH2) and the core antenna/reaction center complex (LH1-RC), play an essential role in the primary process of purple bacterial photosynthesis, that is, capturing light energy, transferring it to the RC where it is used in subsequent charge separation. Establishment of experimental platforms is required to understand the function of the supramolecular assembly of LH2 and LH1-RC molecules into arrays. In this study, we assembled LH2 and LH1-RC arrays into domain-structured planar lipid bilayers placed on a coverglass using stepwise combinations of vesicle-to-planar membrane formation and vesicle fusion methods. First, it was shown that assembly of LH2 and LH1-RC in planar lipid bilayers, through vesicle-to-planar membrane formation, could be confirmed by absorption spectroscopy and high resolution atomic force microscopy (AFM). Second, formation of a planar membrane incorporating LH2 molecules made by the vesicle fusion method was corroborated by AFM together with quantitative analysis by surface plasmon resonance (SPR). By combining planar membrane formation and vesicle fusion, in a stepwise manner, LH2 and LH1-RC were successfully organized in the domain-structured planar lipid membrane. This methodology for construction of LH2/LH1-RC assemblies will be a useful experimental platform with which to investigate energy transfer from LH2 to LH1-RC where the relative arrangement of these two complexes can be controlled.

In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and the light harvesting-reaction center complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a molecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. To address this issue, direct observation of the assembly at the molecular level is necessary to analyze its function. In this study, we reconstituted photosynthetic membrane proteins into artificial lipid bilayers and directly observed their assembly by AFM. The absorption spectra of the reconstituted proteins showed characteristic Qy bands of bacteriochlorophyll a that were identical to those of intact proteins. AFM observation of the reconstituted membranes revealed that LH2 and LH1-RC were successfully assembled into the lipid bilayer, and their observed structures were in good agreement with corresponding crystallographic structures. Specifically, binary proteins, i.e., LH2/LH1-RC and LH2/LH1, which form a densely packed molecular assembly, could be clearly identified at the molecular level by this method of observation. Energy transfer from LH2 to LH1-RC in a reconstituted lipid bilayer was observed by steady-state fluorescence spectroscopy. Enhanced energy transfer was confirmed in the membrane phase compared to that in a homogeneous micellar solution. Such reconstituted molecular assemblies are useful experimental platforms to investigate the relationship between supramolecular arrays and function. [DOI: 10.1380/ejssnt.2011.15]

Previously we developed dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) for use in small interfering RNA (siRNA) therapy. In the present study, mammalian target of rapamycin (mTOR) expression in cancer cells was silenced with mTOR-siRNA (simTOR) formulated in TEPA-PCL modified with Ala-Pro-Arg-Pro-Gly (APRPG), a peptide having affinity for vascular endothelial growth factor receptor-1 (VEGFR-1). We investigated the effects of inhibition of mTOR, focusing on the differences between cells treated with simTOR and those with rapamycin in terms of Akt (ser473) phosphorylation and antiproliferative effects. Rapamycin treatment is known to induce Akt (ser473) phosphorylation which attenuates the antiproliferative effects of rapamycin. As a result, knockdown of mTOR did not alter or only slightly reduced Akt (ser473) phosphorylation in phosphatase and tensin homolog deleted from chromosome 10 (PTEN)-null (LNCaP and MDA-MB-468 cells) and PTEN-positive (DU 145 and MDA-MB-231) cells, although rapamycin induced Akt (ser473) phosphorylation of these cells. Rapamycin suppressed the growth of PTEN-null cells, in which the rapamycin-sensitive mTOR complex 1 (mTORC1) is excessively activated. On the other hand, rapamycin did not suppress the growth of PTEN-positive cells possibly through a negative feedback mechanism via the rapamycin-insensitive mTOR complex 2 (mTORC2) signaling pathway. In contrast, simTOR significantly suppressed the growth of cancer cells regardless of the presence of PTEN, possibly through inhibition of both mTORC1 and mTORC2. These results indicate that mTOR knockdown using APRPG-TEPA-PCL/simTOR is likely to be an effective strategy for cancer siRNA therapy.

Molecular assembly of Zn-porphyrin pigments on a gold electrode using synthetic 1α-helix hydrophobic polypeptides which have similar amino acid sequences to the hydrophobic core in the native photosynthetic light-harvesting (LH) 1-β polypeptide from Rhodobacter sphaeroides has been achieved. This method is clearly successful in allowing assembly of porphyrins together with LH1 type functional complexes with a defined distance and orientation on the electrode. In this case, the photocurrent direction and the distance of electron transfer of pigments could be controlled by these synthetic LH1 model polypeptides. This method will be useful for the self-assembly of these pigment and protein complexes in order to study the energy transfer and electron transfer reactions between individual pigments in the supramolecular complexes on the electrode, as well as to provide insight into the effect of the distance and orientation of pigments and the effect of the structure of 1α-helix hydrophobic polypeptide on the energy transfer and electron transfer reactions.

Synthetic cationic lipids are promising transfection agents for gene therapy. We report here that polyamine conjugates of dialkyl phosphates, combined with natural lipids and assembled in the form of liposomes (polycationic liposome: PCL), possess high transfection activity in the COS-1 cell line. Furthermore, we describe the functional morphology of the PCL/DNA complexes as revealed by atomic force microscopy (AFM). The conjugates were synthesized from dialkyl phosphates (with alkyl chain lengths of 12, 14, or 16 carbons) by reaction with the polyamine molecules, spermidine, spermine, or polyethylenimine (PEI(1800)). [Dewa, T., et al. Bioconjugate Chem. 2004, 15, 824]. The PCL composed of the spermidine and C16 conjugate combined with phospholipid and cholesterol (conjugate/phospholipid/cholesterol = 1/1/1 as a molar ratio) exhibited 3.6 times higher activity than that of a popular commercial product. Systematic tests revealed clear correlations of the transgene activity with physical properties of the polyamine, in particular, that longer alkyl chains and the lower molecular weight polyamines (spermidine, spermine) favor high efficacy at the higher nitrogen/phosphate ratio = 24 (N/P, stoichiometric ratio of nitrogen in the conjugate to phosphate in DNA). The low molecular weight polyamine-based PCLs, which formed 150-400 nm particles with plasmid DNA (lipoplexes), exhibited approximately 3-fold higher gene transfer activity than micellar aggregates (lacking phospholipid and cholesterol) of the corresponding conjugate. In contrast, the PEI-based PCL formed large aggregates (approximately 1 microm), that, like the micellar aggregate form, had low activity. Activity of the low molecular weight polyamine-based PCLs increased linearly with the N/P of the lipoplex up to N/P = 24. Formation of lipoplexes was examined by agarose gel electrophoresis, dynamic light scattering (DLS), and AFM. At the lower N/P = 5, large aggregates of complex (approximately 1 microm), in which DNA molecules were loosely packed, were observed. At higher N/P, lipoplexes were converted into smaller particles (150-400 nm) having a lamellar structure, in which DNA molecules were tightly packed. Such morphological features of the lipoplex correlate with the dependence of transfection on the N/P in that the lamellar structures gave superior transfection. AFM also indicated that the lipoplexes disassembled significantly, releasing DNA, when the lipoplexes were exposed to acidic conditions (pH 4). The significance for transfection activity of the metamorphosis of bilayer lipoplexes is discussed relative to that of the less active micellar aggregate form, which is unresponsive to pH change.

光合成膜中では，光エネルギー変換機能をもつタンパク質超分子複合体に含まれる色素分子が，タンパク質と相互作用をしてその機能を発揮している．本報では，光合成のアンテナ系タンパク質超分子複合体(LH1 複合体)中のカロテノイド色素分子の構造と機能の関係を調べるために，共役二重結合鎖長の異なるカロテノイドを用いて LH1 複合体の再構成を行った．そして，得られた再構成 LH1 複合体の Stark スペクトル測定を行い，カロテノイドおよびバクテリオクロロフィル a(BChl a)周辺の静電場環境を評価した．さらに，カロテノイド分子の半経験的分子軌道計算を行って，LH1 複合体中のカロテノイド分子の立体構造を解析した．その結果，このカロテノイド分子は，結晶構造が報告されている LH2 複合体中のカロテノイドと同様に，全トランス体のねじれ構造でタンパク質との結合部位に組織化されていると考えられた．

Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.

Phospholipid-linked naphthoquinones separated by spacer methylene groups (C n ), PE-C n -NQ ( n = 0, 5, 11), were synthesized to investigate the quinone-mediated electron transfers on a glassy carbon (GC) electrode covered with phospholipids membrane. The PE-C n -NQ could be incorporated in lipid bilayer composed of phosphatidylcholine and exhibited characteristic absorption spectral change corresponding to their redox state, quinone/hydroquinone. The cyclic voltammogram of PE-C n -NQ-containing lipid bilayer modified on a GC electrode indicated a set of waves corresponding to the consecutive two-electron and two-proton transfer reduction of the quinone moiety. The peak currents of PE-C n -NQ as a function of temperature showed a sharp break point in the current–temperature behavior, reflecting the gel-fluid phase transition. The shape of the cyclic voltammograms changed with the pH of the buffer solution. Below pH 6 the first step of the reduction of quinone was a monoprotonation of quinone, whereas above pH 10 the first step of the oxidation was a monodeprotonation of hydroquinone. This indicates that reaction sequences of quinone/hydroquinone were different with the change of the pH. These results showed that the PE-C n -NQ exhibited electron transfer associated with proton transfer in the lipid membranes, depending on the diffusivity of the redox species in the membrane and pH. Interestingly, less effect of the number of methylene of the spacer group on the peak currents was observed. Comparison of manganese porphyrin-mediated electron transfer that depends on the spacer methylene lengths [M. Nango, T. Hikita, T. Nakano, T. Yamada, M. Nagata, Y. Kurono, T. Ohtsuka, Langmuir 14 (1998) 407] is made.

Molecular assembly of Zn porphyrin pigments, Zn substituted bacteriochlorophyll a ([Zn]-BChl a ) and Zn mesoporphyrin monomer (ZnMPMME) on a gold electrode using synthetic 1α-helix hydrophobic polypeptides which have similar amino acid sequences to the hydrophobic core in the native photosynthetic light-harvesting (LH) 1-β polypeptide from Rhodobacter sphaeroides , has been achieved: this process is dependent on the structures of pigments and polypeptides. Interestingly, an enhanced photoelectric current was observed when ZnMPMME with the LH1 model polypeptide in an α-helical configuration was assembled onto the electrode.

We reported here that polyethylene glycol (PEG)-linked manganese pyrochlorophyllide a (PEG-MnPChlide a) possesses remarkable catalytic activity comparable to horseradish peroxidase (HRP). The PEG-MnPChlide a catalyzed the oxidation decoloration reaction of C.I. Acid Orange 7 by hydrogen peroxide under a mild aqueous condition, pH 8.0 at 25 degrees C. The manganese pyrochlorophyride a methylester (MnPChlide a ME) dissolved in a Triton X-100 micellar solution also exhibited the catalytic activity, indicating the micellar environment plays an important role in the catalytic reaction. The reaction rate was accelerated by addition of imidazole. The catalytic reactions were analyzed by Michaelis-Menten kinetics, revealing that the higher reactivity of catalyst-substrate complex is responsible for the present catalytic reaction system.

Bacterial photosynthetic membrane proteins, light-harvesting antenna complex (LH1), reaction center (RC), and their combined 'core' complex (LH1-RC) are functional elements in the primary photosynthetic events, i.e., capturing and transferring light energy and subsequent charge separation. These photosynthetic units (PSUs) isolated from Rhodospirillum rubrum (Rs. rubrum) were assembled onto an ITO electrode modified with 3-aminopropyltriethoxysilane (APS-ITO). The near IR absorption spectra of PSUs on the assembled electrodes were identical to those of solutions, indicating that the LH1 and LH1-RC core complexes were native on the electrode. Photocurrent response of PSUs on the electrode was examined upon illumination of the LH1 complex at 880 nm. The LH1-RC and a mixed assembly of LH1 and RC exhibited photocurrent response, but not LH1 only, consistent with the function of these PSUs, capturing light energy and transferring electron. This result provides useful methodology for building an artificial fabrication of PSUs on the electrode.

Small interfering RNA (siRNA) has the potential to be a candidate as a cure for intractable diseases. However, an appropriate vector is required for siRNA delivery because of the low transfection efficiency of siRNA without a vector and its easy degradation in vivo. Here, we report a simple, only one step, and efficient method for siRNA encapsulation into a lipidic nanocarrier by freeze-thawing: siRNA was entrapped between the lipid layers of multi-layer liposomes by freeze-thawing of lipoplexes composed of polycation liposomes (PCLs) and siRNA. siRNA-holding capacity to the PCL was increased by repeating freeze-thaw of the lipoplex up to 5cycles. Although siRNA in the conventional lipoplex was degraded after incubation in 90% fetal bovine serum for 72h, siRNA in the frozen and thawed lipoplex was not degraded. Interestingly, we found that the lipoplex formed a "packed multi-layer" structure after the freeze-thawing of "single-layer" PCL and siRNA complex, suggesting that siRNA exists between the lipid layers working as a binder. The frozen and thawed lipoplex showed significantly higher knockdown efficacy compared with the conventional lipoplex. In addition, PEGylated freeze-thawed lipoplexes delivered a higher amount of siRNA to a tumor in vivo compared with the PEGylated conventional ones. These results provide an attractive strategy for "one-step" encapsulation of siRNA into liposomes by freeze-thawing.

To reveal the structure-function relationship of membrane proteins, a membrane environment is often used to establish a suitable platform for assembly, functioning, and measurements. The control of the orientation of membrane proteins is the main challenge. In this study, the electron conductivity and photocurrent of a light-harvesting/reaction center core complex (LH1-RC) embedded in a lipid membrane were measured using conductive atomic force microscopy (C-AFM) and photoelectrochemical analysis. AFM topographs showed that LH1-RC molecules were well-orientated, with their H-subunits toward the membrane surface. Rectified conductivity was observed in LH1-RC under precise control of the applied force on the probe electrode (

The KcsA potassium channel is a prototypical channel of bacterial origin, and the mechanism underlying the pH-dependent gating has been studied extensively. With the high-resolution atomic force microscopy (AFM), we have resolved functional open and closed gates of the KcsA channel under the membrane-embedded condition. Here we surprisingly found that the pH-dependent gating of the KcsA channels was associated with clustering-dispersion dynamics. At neutral pH, the resting, closed channels were coalesced, forming nanoclusters. At acidic pH, the open-gated channels were dispersed as singly isolated channels. Time-lapse AFM revealed reversible clustering-dispersion transitions upon pH changes. At acidic equilibrium, a small fraction of the channels was nanoclustered, in which the gate was apparently closed. Thus, it is suggested that opening of the gate and the dispersion are tightly linked. The interplay between the intramolecular conformational change and the supramolecular clustering-dispersion dynamics provides insights into understanding of unprecedented functional cooperativity of channels.

Bacterial photosynthetic antenna complexes are composed of α-helical hydrophobic polypeptides and pigments (e.g., bacteriochlorophyll a). We report here self-assembling properties of an engineered hydrophobic polypeptide with zinc-substituted bacteriochlorophyll a ([Zn]-BChl a) in various lipid bilayer to investigate the effect of lipid species on the self-assembling properties. When the polypeptide and [Zn]-BChl a were mixed in surfactant solution (n-octyl β-D-glucopyranoside: OG) at 25°C, the absorption band [Zn]-BChl a was red-shifted from 770 to 812 nm, that is assignable to quasi-dimeric “subunit-type” complex. By subsequent dilution and cooling of the solution, the absorption band further red-shifted to 836 nm indicative for progressed assembly, ‘LH1-type’ complex. When the subunit-type complex was assembled in lipid bilayer, e.g., phosphatidylcholines (PC’s) and phosphatidylglycerol (PG’s), the assembling property to form LH1-type complex prominently depended on the character of their fatty acid chains in the phospholipids. The subunit complex remained in the fluidic bilayers, but further assembled to form LH1-type complex in solid-like phospholipid bilayers, suggesting that intermolecular force of phospholipid governs the assembling of the polypeptide/[Zn]-BChl a complex.

The subunit light-harvesting 1 (LH 1) complexes isolated from photosynthetic bacteria Rhodospirillum rubrum using n-octyl-beta-glucoside were reassociated and adsorbed on a mica substrate using spin-coat methods with the aim of using this LH complex in a nanodevice. The near-IR absorption and fluorescence spectra of the LH 1 complexes indicated that the LH 1 complex on the mica was stable, and efficient energy transfer from a carotenoid to a bacteriochlorophyll a was observed. Atomic force microscopy of the reassociated LH 1 complexes, under air, showed the expected ringlike structure. The outer and inner diameters of the ringlike structure of the LH 1 complex were approximately 30 and 8 nm, respectively, and the ringlike structure protruded by 0.2-0.6 nm.

We present here a novel approach to organization of an α-helical hydrophobic polypeptide complex in lipid bilayers in a 'lipid-domain-selective' manner. The key strategy is to use disulfide linkage between a cysteine-bearing polypeptide and a disulfide-based phospholipid, the latter of which forms a phase-separated lipid domain in a binary lipid bilayer with an incompatible phospholipid lacking disulfide group. The cross-linkage between the polypeptide and the phospholipid can be expected that the polypeptide acquires a higher affinity to the disulfide-based lipid domain than to the other one, resulting in selective distribution of the polypeptide in the former domain. The polypeptide analogous to a photosynthetic bacterial light-harvesting polypeptide binds a pigment, zinc-substituted bacteriochlorophyll a ([Zn]-BChl a), to form the light-harvesting polypeptide/pigment complex. The complex was incorporated into an incompatible binary lipid bilayer combination either the disulfide-based phospholipids bearing oleoyl chains (DO-DO)/1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/DSPC. The lipid-domain-selective organization was confirmed by fluorescence resonance energy transfer (FRET) from a rhodamine-labeled lipid (Lissamine rhodamine B 1,2-diacyl-sn-glycero-3-phosphoethanolamine: either N-Rh-DOPE or N-Rh-DSPE) to the assembled [Zn]-BChl a. Considering the relative affinity of these fluorescent lipid probes, N-Rh-DOPE and N-Rh-DSPE prefer to fluid and gel domains, respectively, because of the similarity of their acyl chains. When the fluorescent lipid probe, N-Rh-DOPE, was applied to the binary lipid system, DO-DO/DSPC, the higher FRET efficiency was observed compared to the counterpart FRET assay using N-Rh-DSPE. In contrast, for the DOPC/DSPC bilayer, the difference in the FRET efficiency between the fluorescent lipid probe systems was negligible. These results suggest that the polypeptide/pigment complex is preferentially incorporated into the disulfide-based lipid domain. Total internal reflection fluorescence (TIRF) microscopic observation of the assembly also evidenced heterogeneous structure by detection of fluorescence from [Zn]-BChl a. [DOI: 10.1380/ejssnt.2005.145]

We succeeded in preparing a fibrous nanotube of green fluorescent protein by conjugation with poly(2-vinylpyridine) and use of a microfluidic synthesis technique. By inducing a sudden pH change in ...

非ウイルス系遺伝子導入キャリヤーとして, スペルミジン, スペルミン, およびポリエチレンイミン (平均分子量1800) をカチオン部位にもつジパルミトイルポスファチジン酸誘導体を新規に合成した. β-ガラクトシダーゼアッセイにより, これらが遺伝子導入効果を有することがわかった. これらのポリカチオン化リン脂質は水溶液中では90～200nm程度の集合体を形成しており, これらがDNAとの複合体を形成することを原子間力顕微鏡により直接観察した. 遺伝子導入効果は, その複合体サイズが小さいものほど高いことが認められた.

Peptide-based gemini surfactants, called PG-surfactants, consisting of a tri- or tetra-Asp peptide core and acetylated dodecylamine-conjugated Cys residues at both the N- and C-terminal ends (D3C12...

Bacterial photosynthetic antenna polypeptide (LH1-α) was synthesized as a water-soluble fusion protein with maltose-binding protein (MBP) and a His-tag portion (MBP–rubα-YH) using an E. coli expres...

Photoresistance of a pigment-binding membrane protein, light-harvesting 2 (LH2) complex from the photosynthetic bacterium, Rhodopseudomonas acidophila , was investigated by fluorescence of single LH2 complexes at a temperature of 296 K. Before irreversibly stopping fluorescence, a single LH2 complex in phospholipid bilayer of dimyristoylphosphatidylcholine (DMPC) emitted on average 4 times more fluorescence photons than a complex in detergent micelle of octylglucopyranoside (OG). Fluorescence-excitation spectrum of single LH2 complexes taken at 5 K showed that the LH2 complex is structurally less perturbed in DMPC bilayer than in OG micelle, suggesting that structural instability reduces photoresistance of LH2.

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.

When immersed in an aqueous solution of alcohol or alkyl acetate as a guest, 9,10-bis(3,5-dihydroxyphenyl)anthracene 1 as a hydrogen-bonded solid host incorporates two mols of the guest together with 5—8 mols of water. The binding isotherm shows a sharp threshold guest concentration characteristic of guest-induced phase-transition. The threshold concentrations as a measure of affinities are highly dependent on the hydrophobicities of the guests. Host 1 also catalyzes the Diels-Alder reaction of acrylaldehyde and 1,3-cyclohexadiene in an aqueous environment. The preserved 1 : 2 (host-to-guest) stoichiometry, spectroscopic evidence (ΔνC=O), guest-desorption behaviors, and X-ray powder-diffraction studies indicate that host-guest hydrogen-bonding in collaboration with apolar interactions is responsible for the selective guest-binding and catalysis under the present aqueous conditions.

Treatment of anthracenebisresorcinol (1·4H) with La(OiPr)3 and binaphthol (BN·2H) as a chiral ligand affords an amorphous and insoluble 1:2:2 adduct (chiral La host) formulated as (1·2H)2-·2(LaBN)+...

The nearest-neighbor recognition method is applied to the problem of phospholipid clustering in bilayers composed of di[1,2-dimyristoyl-sn-glycero-3-phosphoethanol(3‘-thio)propionamide] (I), di[1,2-distearoyl-sn-glycero-3-phosphoethanol(3‘-thio)propionamide] (II), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanol(3‘-thio)propionamide−1,2-distearoyl-sn-glycero-3-phosphoethanol(3‘-thio)propionamide (III). The value and the composition dependency of the apparent equilibrium constant, K, defined by the equilibrium concentrations of homodimers (I and II) and the corresponding heterodimer (III), allow one to distinguish among three fundamentally different classes of clustering: (i) random clustering, (ii) cooperative clustering, and (iii) nonrandom−noncooperative clustering. Experimental results indicate that random clustering of these phospholipids is pervasive in fluid bilayers, whereas cooperative clustering exists in the gel−fluid coexistence region. In the physiologically relevant fluid phase, these same li...

Treatment of anthracenebisresorcinol 1 (a tetraol) with Al(CH3)2Cl or Al(OiPr)3 affords an amorphous solid formulated as 14−·2[AlCl]2+ or 14−·2[AlOiPr]2+ (14− is the deprotonated tetraanionic species of host 1). These solids (AlCl and AlOiPr hosts) as recoverable and reusable Lewis acids catalyze the Diels–Alder reaction of acrolein with 1,3-cyclohexadiene, where the former (AlCl) at 3 mol% exhibits a much higher catalytic activity with a halflife of the reaction of τ=1 min than the latter (AlOiPr) with τ=80 min. The efficient solid-state catalysis by the AlCl host with a turnover rate of 30/min is based on the combination of space vacancy (microporosity) and coordination vacancy (coordinative unsaturation) as revealed by 27Al MAS NMR spectroscopy.

The solid/gas complexation of anthracenebisresorcinol host (1) and ethyl acetate as a guest was monitored by pressure-decay, X-ray powder diffraction, and gravimetric/calorimetric thermal desorption analyses. It involves an exothermic (∼30 kcal/mol) phase transition and exhibits “vertical” adsorption as well as desorption “isotherms” at a threshold (equilibrium) pressure (Pth) of the guest vapor. This is in accord with the phase rule for a two-component/three-phase/one-freedom system. The activation energies of adsorption and desorption are 2.3 and 34 kcal/mol, respectively; desorption at 25 °C takes weeks. The crystal structure of the ethyl acetate adduct illustrates how the flexible hydrogen-bonded network of host 1 adjusts itself to the small guest. Other polar guests (ester, ketone, and alcohol) behave similarly and give rise to desorption-resistant stable adducts, except for the least bulky members of the ester and ketone guests, i.e., methyl acetate and acetone. The mechanism of phase transition is ...

The mixing behavior of phosphoethanolamine (PE)-like and phosphoglycerol (PG)-like lipids have been examined in the fluid bilayer state by use of nearest-neighbor recognition methods [Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1995, 117, 7838]. Disulfide-based phospholipid dimers derived from 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) have been synthesized, containing PE- and PG-like head groups. Subsequent exchange and equilibration of the monomer units, via thiolate−disulfide interchange, produced a nonstatistical distribution of dimers in the fluid bilayer phase (60 °C); i.e., a heterodimer/homodimer ratio of 1.76 ± 0.05 was formed. Analogous membranes that were rich in cholesterol (i.e., 29 mol %) produced a statistical mixture of dimers. Equilibration experiments that were carried out using PE-like and PG-like lipids derived from 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and DMPE, respectively, gave similar results, except that a greater level of nearest-neigh...

Previously, we developed tetraethylenepentamine-based polycation liposomes (TEPA-PCL) as a vector for the delivery of small RNAs. In the present research, we attempted tumor-targeted delivery of miR-499 via systemic administration and evaluated the potency of this system as a therapeutic strategy to treat cancer. Lipoplexes were formed by mixing cholesterol-grafted miR-499 (miR-499-C) with TEPA-PCL. Firstly, human umbilical endothelial cells (HUVECs) and Colon 26 NL-17 mouse carcinoma cells were transfected with these lipoplexes in vitro. The results showed that miR-499 had antiangiogenic effects on the HUVECs and suppressed the secretion of vascular endothelial growth factor (VEGF) from the Colon 26 NL-17 cells. In addition, the growth of the latter cells was inhibited by transfection with miR-499-C/TEPA-PCL. For in vivo delivery of miR-499 to tumors via systemic injection, miR-499-C/TEPA-PCL were decorated with Ala-Pro-Arg-Pro-Gly (APRPG) peptide-conjugated polyethylene glycol (PEG) to prepare APRPG-PEG-modified lipoplexes carrying miR-499 (APRPG-miR-499). APRPG-miR-499 were injected into tumor-bearing mice via a tail vein, and these lipoplexes accumulated sufficiently in both angiogenic vessels and cancer cells. In addition, the expression of miR-499-target proteins and VEGF in the tumor cells was clearly suppressed by the treatment with APRPG-miR-499. Finally, the therapeutic effect of miR-499 on tumor growth was evaluated in mice. The tumor growth was significantly inhibited by the intravenous injection of APRPG-miR-499 at such a low dose as 0.5mg/kg. These results suggest that miR-499 delivered by the present system has excellent potency to treat cancer via integrative anticancer actions.

We designed novel peptide gemini surfactants (PG-surfactants), DKDKC12K and DKDKC12D, which can solubilize Photosystem I (PSI) of Thermosynecoccus elongatus and Photosystem II (PSII) of Thermosynecoccus vulcanus in an aqueous buffer solution. To assess the detailed effects of PG-surfactants on the original supramolecular membrane protein complexes and functions of PSI and PSII, we applied the surfactant exchange method to the isolated PSI and PSII. Spectroscopic properties, light-induced electron transfer activity, and dynamic light scattering measurements showed that PSI and PSII could be solubilized not only with retention of the original supramolecular protein complexes and functions but also without forming aggregates. Furthermore, measurement of the lifetime of light-induced charge-separation state in PSI revealed that both surfactants, especially DKDKC12D, displayed slight improvement against thermal denaturation below 60 °C compared with that using β-DDM. This degree of improvement in thermal resistance still seems low, implying that the peptide moieties did not interact directly with membrane protein surfaces. By conjugating an electron mediator such as methyl viologen (MV(2+)) to DKDKC12K (denoted MV-DKDKC12K), we obtained derivatives that can trap the generated reductive electrons from the light-irradiated PSI. After immobilization onto an indium tin oxide electrode, a cathodic photocurrent from the electrode to the PSI/MV-DKDKC12K conjugate was observed in response to the interval of light irradiation. These findings indicate that the PG-surfactants DKDKC12K and DKDKC12D provide not only a new class of solubilization surfactants but also insights into designing other derivatives that confer new functions on PSI and PSII.

We designed novel bilayer-forming amphiphiles based on the cyclic oligo-Asp-based peptide gemini (PG) surfactants cr-D2C12 and cr-D3C12, which consist of -Cys(Asp)nCys- (n = 2 or 3) as a core peptide and two Cys residues containing a dodecylamidomethyl group. Dynamic light scattering and transmission electron microscopy measurements revealed the formation of spherical bilayer membranes that could incorporate the light-harvesting antenna complex 2 (LH2) from Rhodopseudomonas acidophila . Furthermore, this proteoliposome-like conjugate could be assembled onto cationized glass and mica to form planar bilayer membranes incorporating LH2. Using atomic force microscopy, we observed LH2 protruding (ca. 1.2-1.5 nm) from flat terraces of the planar bilayer membranes formed from cr-D2C12 or cr-D3C12. Thus, our designed PG surfactants are a new class of bilayer-forming amphiphiles that may be applied to the study of various membrane proteins.

We previously developed a microRNA (miRNA) delivery system by using dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL), applied it to miR-92a delivery, and demonstrated its gene-silencing potential and effective anti-angiogenic effects. In the present study, we investigated the mechanism of intracellular delivery of cholesterol-grafted miR-92a (miR-92a-C) into cells.To investigate the intracellular distribution of miR-92a-C/TEPA-PCL complex, we used human umbilical vein endothelial cells and examined certain points after transfection: (i) the time-course of miR-92a-uptake into the cells; (ii) the endocytosis pathway induced by miR-92a-C/TEPA-PCL; (iii) the capability of miR-92a-C/TEPA-PCL to escape from the endosomes; and (iv) the release of miR-92a-C from TEPA-PCL in the cytoplasm.Our data indicated that miR-92a-C formulated in TEPA-PCL accumulated in and was spread throughout the cytoplasm in a time-dependent manner, and was taken up into the cells by macropinosome-mediated endocytosis. In addition, the surface charge of miR-92a-C/TEPA-PCL was neutral at pH 7.4 and was charged positively at around pH 5.5, which is the inner pH of endosomes. When the late endosomes/lysosomes were stained with Lysotracker, miR-92a-C/TEPA-PCL efficiently escaped from the endosomes into the cytoplasm, possibly through the proton-sponge effect. Furthermore, miR-92a-C spread throughout whole cytoplasm and did not co-localize completely with TEPA-PCL, indicating that some of the miR-92a-C was present in free form in the cytoplasm.The results of the present study suggest that TEPA-PCL-based lipoplexes have an excellent potential to deliver microRNAs into the cytoplasm of cells and to induce RNA silencing action mediated by microRNAs and other small RNAs.

Novel polycation liposomes decorated with cyclic(Cys-Arg-Gly-Asp-D-Phe) peptide (cyclicRGD)-polyethylene glycol (PEG) (RGD-PEG-polycation liposomes (PCL)) were previously developed for cancer therapy based on RNA interference. Here, we demonstrate the in vivo delivery of small interfering RNA (siRNA) to tumors by use of RGD-PEG-PCL in B16F10 melanoma-bearing mice. Pharmacokinetic data obtained by positron emission tomography showed that cholesterol-conjugated siRNA formulated in RGD-PEG-PCL markedly accumulated in the tumors. Delivered by RGD-PEG-PCL, a therapeutic cocktail of siRNAs composed of cholesterol-conjugated siRNAs for c-myc, MDM2, and vascular endothelial growth factor (VEGF) were able to significantly inhibit the growth of B16F10 melanoma both in vitro and in vivo. These data suggest that targeted delivery of siRNAs by use of RGD-PEG-PCL has considerable potential for cancer treatment.

Evidence has been obtained for the existence of lateral heterogeneity in fluid bilayers composed of mixtures of saturated and unsaturated phospholipids by use of nearest-neighbor recognition (NNR) ...

The mixing behavior of three saturated ester phospholipids with one ether analog has been examined in the bilayer state by use of nearest-neighbor recognition methods [Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1995, 117, 7838]. Specifically, the miscibility of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) with 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) has been investigated by analyzing mixtures of corresponding dithidipropionyl-based dimers that have been chemically equilibrated via thiolate−disulfide interchange reactions. In the fluid phase, those membranes derived from DMPE plus DHPE were found to exhibit lateral heterogeneity. In contrast, fluid membranes that were derived from DPPE plus DHPE, and also DSPE plus DHPE, were randomly arranged. Inclusion of cholesterol did not significantly alter the lateral distribution of any of these phospholipid m...

RNA interference has received much attention as a novel therapeutic strategy. MicroRNA (miRNA) appears to be promising as a novel nucleic-acid medicine because it is able to suppress a series of protein expression that relates to a specific event such as angiogenesis. In the present study, we used dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) as a delivery system for miR-92a, one of the miRNAs regulating angiogenesis, and attempted to deliver miR-92a to angiogenic endothelial cells for the development of cancer therapy by anti-angiogenesis.Cholesterol-grafted miR-92a (miR-92a-C) was bound to TEPA-PCL, and the ratio of nitrogen of TEPA-PCL to phosphorus of miR-92a-C (N/P ratio) was optimized. This complex was transfected into human umbilical vein endothelial cells (HUVECs), and the intracellular localization of miR-92a-C was observed under a confocal laser-scanning microscope by the use of fluorescein isothiocyanate-labeled miR-92a-C. After transfection of HUVECs with miR-92a-C/TEPA-PCL, the expression of miR-92a-target proteins (e.g. integrin α5, mitogen-activated protein kinase kinase 4, sphingosine-1-phosphate receptor 1) was examined by western blotting, and a tube formation assay was performed.The complex of miR-92a-C with TEPA-PCL was formed and miR-92a-C remained stable with TEPA-PCL at the N/P ratio of 10. After transfection of HUVECs with miR-92a-C complex, miR-92a-C spread into the whole cytoplasm of the cells without any change of cellular morphology, and the expression of several proteins encoded by miR-92a-target genes was suppressed. Furthermore, the capability of forming capillary tubes was impaired in complex-treated HUVECs.We have developed a miR-92a delivery system into angiogenic endothelial cells by the use of TEPA-PCL. These results suggest that miR-92a-C/TEPA-PCL is promising for the treatment of tumors via the suppression of angiogenesis.

A polyhistidine (His) tag was fused to the C- or N-terminus of the light-harvesting (LH1)-α chain of the photosynthetic antenna core complex (LH1-RC) from Rhodobacter sphaeroides to allow immobilization of the complex on a solid substrate with defined orientation. His-tagged LH1-RCs were adsorbed onto a gold electrode modified with Ni-NTA. The LH1-RC with the C-terminal His-tag (C-His LH1-RC) on the modified electrode produced a photovoltaic response upon illumination. Electron transfer is unidirectional within the RC and starts when the bacteriochlorophyll a dimer in the RC is activated by light absorbed by LH1. The LH1-RC with the N-terminal His-tag (N-His LH1-RC) produced very little or no photocurrent upon illumination at any wavelength. The conductivity of the His-tagged LH1-RC was measured with point-contact current imaging atomic force microscopy, indicating that 60% of the C-His LH1-RC are correctly oriented (N-His 63%). The oriented C-His LH1-RC or N-His LH1-RC showed semiconductive behavior, that is, had the opposite orientation. These results indicate that the His-tag successfully controlled the orientation of the RC on the solid substrate, and that the RC produced photocurrent depending upon the orientation on the electrode.

Poly(γ-methyl L-glutamate) (PMLG) membrane containing manganese tetrakis(pentafluorophenyl)porphyrin1 (MnPFPP) has been studied for the transmembrane redox reaction between dithionite and ferricyanide in separated solutions. The effects of the ligand species, namely, OH−, Cl−, imidazole (Im), pyrazine (Pyz), triethylamine (TEA), and pyridine (Py), on the electron transfer rate in the membrane were examined. UV/VIS spectroscopy and sorption measurements confirm that these ligands are sorbed by the membrane from the external buffer solution and coordinate to MnPFPP. With the membrane containing 160 μmol · g−1 of MnPFPP, the apparent rate factor, estimated as the pseudo-first-order rate constant of the bleach of ferricyanide times membrane thickness, follows the order Im ≫ Cl− > Pyz > TEA, OH−, Py > 0, suggesting that Im, Cl− and Pyz are effective ligands for the electron transfer. Such a ligand-specific transfer stems from the electron-transferability of the ligands between MnPFPP's.

Transmembrane electron transfer across S-cyanoethylated keratin (SCEK) and liposomal membranes catalyzed by (1), MnTTP-(CH2)n-MnP(COOMe)3, n = 2, 3, 12 showed that an enhanced electron transfer was observed especially when n = 2 or 3 on (1) in these membranes and imidazole was present.