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The affinity of a synthetic polymer nanoparticle (NP) to a target biomacromolecule is determined by the association and dissociation rate constants (kon, koff) of the interaction. The individual rates and their sensitivity to local environmental influences are important factors for the on-demand capture and release a target biomacromolecule. Positively charged NPs for small interfering RNA (siRNA) delivery is a case in point. The knockdown efficacy of siRNA can be strongly influenced by the binding kinetics to the NP. Here, we show that kon and koff of siRNA to NPs can be individually engineered by tuning the chemical structure and composition of the NP. N-Isopropylacrylamide-based NPs functionalized with hydrophobic and amine monomers were used. koff decreased by increasing the amount of amine groups in the NP, whereas kon did not change. Importantly, NPs showing a low koff at pH 5.5 together with a high koff at pH 7.4 showed high knockdown efficiency when NP/siRNA complexes were packaged in lipid nanoparticles. These results provide direct evidence for the premise that the efficacy of an siRNA delivery vector is linked with the strong affinity to the siRNA in the endosome and low affinity in the cytoplasm.

Phycobilisomes (PBSs) are photosynthetic antenna megacomplexes comprised of pigment-binding proteins (cores and rods) joined with linker proteins. A rod-type PBS that does not have a core is connected to photosystem I (PSI) by a pigment-free CpcL linker protein, which induces a red-shift of the absorption band of phycocyanobilin (PCB) in the rod (red-PCB). Herein, the isolated supercomplex of the rod-type PBS and the PSI tetramer from Anabaena sp. PCC 7120 were probed by picosecond laser spectroscopy at 77 K and by decay-associated spectral analysis to show that red-PCB mediates the fast (time constant = 90 ps) and efficient (efficiency = 95%) transfer of excitation energy from PCB in rod to chlorophyll a (Chl a) in PSI. According to the Förster energy transfer mechanism, this high efficiency corresponds to a 4-nm distance between red-PCB and Chl a, suggesting that β-84 PCB in rod acts as red-PCB.TOC GRAPHIC

While the influence of pKa provided by amine-containing materials in siRNA delivery vectors for use in gene-silencing has been widely studied, there are little reports in which amine pKa is controlled rigorously by using bioisosteres and its effect on gene-silencing. Here, we report that amine pKa could be rigorously controlled by replacement of hydrogen atom(s) with fluorine atom(s). A series of mono- and di-amine lipids with a different number of fluorine atoms were synthesized. The pKa of the polyamine lipids was shifted to a lower value with an increase in the number of fluorine atoms. The optimal pKa for high gene-silencing efficiency varied according to the number of amine residues in the polyamine lipid. Whereas the endosomal escape ability of mono-amine lipid-containing lipid vesicles (LVs) depended on the pKa, that of all tested di-amine lipid-containing LVs showed equal membrane-destabilizing activity. LVs showing moderately weak interactions with siRNA facilitated cytoplasmic release of siRNA, resulting in strong gene-silencing. These findings indicate that appropriate amine pKa engineering depending on the number of amines is important for the induction of effective RNA interference.

Enhancing the light-harvesting potential of antenna components in a system of solar energy conversion is an important topic in the field of artificial photosynthesis. We constructed a biohybrid light-harvesting complex 2 (LH2) engineered from Rhodobacter sphaeroides IL106 strain. An artificial fluorophore Alexa Fluor 647 maleimide (A647) was attached to the LH2 bearing cysteine residue at the N-terminal region (LH2-NC) near B800 bacteriochlorophyll a (BChl) assembly. The A647-attached LH2-NC conjugate (LH2-NC-A647) preserved the integrity of the intrinsic chromophores, B800- and B850-BChls, and carotenoids. Femtosecond transient absorption spectroscopy revealed that the sequential energy transfer A647 → B800 → B850 occurs at time scale of 9–10 ps with monoexponential dynamics in micellar and lipid bilayer systems. A B800-removed conjugate (LH2-NC[B800(−)]-A647) exhibited a significant decrease in energy transfer efficiency in the micellar system; however, surprisingly, direct energy transfer from A647 to B850 was observed at a rate comparable to that for LH2-NC-A647. This result implies that the energy transfer pathway is modified after B800 removal. The results obtained suggested that a LH2 complex is a potential platform for construction of biohybrid light-harvesting materials with simple energy transfer dynamics through the site-selective attachment of the external antennae and the modifiable energy-funnelling pathway.

Lipid-based nanoparticles, a potential nonviral vector due to their good biocompatibility and biodegradability, have been extensively developed for the delivery of small interfering RNA (siRNA). We designed a unique pH-responsive lipid derivative, a dioleylphosphate-diethylenetriamine conjugate (DOP-DETA). DOP-DETA consists of a pH-responsive triamine and unsaturated fatty acids that accelerate membrane fusion. Our results showed that DOP-DETA-based liposomes (DL) efficiently delivered siRNA into the cytoplasm and induced RNA interference even at a low siRNA concentration. The knockdown efficiency of DL depended on the molar ratio of total DL lipids to siRNA. When siRNA was formulated with a sufficient amount of DL, it was efficiently taken up by cells and induced effective gene silencing. Time-lapse imaging showed that siRNA transfected with DL was rapidly internalized into the cells and uniformly dispersed in the cytoplasm within a few minites. The results also showed that DL induced sufficient change in surface charge to allow it to interact with the cell membrane and to allow for rapid endosomal escape. Uptake pathway and time-lapse imaging studies revealed that siRNA was delivered by DL into the cytoplasm, possibly through both macropinocytosis and membrane fusion. The present results emphasize that the modulation of surface charge on nanoparticles is crucial for each siRNA delivery process. Our results also suggest that DL is a potentially useful vector for inducing gene silencing with low-doses of siRNA.

To examine the applicability of fluorinated membrane-forming phospholipids to reconstitution matrices for functional membrane proteins, the membrane properties of a synthetic ether-type phosphatidylcholine (PC) bearing partially fluorinated C18-monoacetylenic (9-octadecynyl) chains, DF8CCH8PC, were compared with those of its non-fluorinated counterpart, DH8CCH8PC. Light-harvesting complex 2 (LH2) and the light-harvesting 1‒reaction center core complex (LH1-RC) isolated from purple photosynthetic bacteria were employed as probe membrane proteins to evaluate the extent to which their reconstitution into DF8CCH8PC membranes could proceed. DF8CCH8PC formed more expanded and more stable fluid monolayers than DH8CCH8PC at the air-water interface at 25 °C; the former PC molecule occupied an area of ca. 0.70 nm2 at a collapse pressure, πc, of 52 mN/m, while the latter occupied an area of ca. 0.55 nm2 at a πc of 45 mN/m. In contrast, the molecular motion detected using fluorescent probes was much more restricted in DF8CCH8PC bilayers than in DH8CCH8PC ones. Although the reconstitution efficiencies of both LH2 and LH1-RC into DF8CCH8PC bilayers were lower than those into DH8CCH8PC bilayers, the membrane proteins incorporated into DF8CCH8PC bilayers showed increased thermostability. The increased thermostability of these proteins in fluorinated PC membranes might be due to the restricted molecular motion in the hydrophobic chains. The results of this study suggest that partially fluorinated PCs can be useful materials for the construction of lipid‒functional membrane protein assemblies including large membrane protein complexes, such as LH1-RC, for biotechnological applications.

Appropriate experimental platforms are required to clarify the structure–function relationships of membrane protein assemblies. In photosynthetic bacteria, light-harvesting complex 2 and light-harvesting/reaction center core complex play key roles in capturing and transferring light energy and facilitating subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly in the photosynthetic membrane. However, the mechanism through which this assembly influences the efficiency of energy conversion remains to be clarified. We review our recent studies that were conducted to evaluate the structure–function relationship of the supramolecular assembly of photosynthetic antenna complexes in various lipid bilayer systems, as well as the construction of novel systems of planar lipid membranes for use as experimental platforms.

The development of additional extraction surfactants for membrane proteins is necessary for membrane protein research, since optimal combinations for the successful extraction of target membrane proteins from biological membranes that minimize protein denaturation are hard to predict. In particular, those that have a unique basal molecular framework are quite attractive and highly desired in this research field. In this study, we successfully constructed a new extraction surfactant for membrane proteins, NPDGC12KK, from the peptide-gemini-surfactant (PG-surfactant) molecular framework. The PG-surfactant is a U-shaped lipopeptide scaffold, consisting of a short linker peptide (-X-) between two long alkyl-chain-modified Cys residues and a peripheral peptide (Y-) at the N-terminal side of long alkyl-chain-modified Cys residues. Using photosystem I (PSI) and photosystem II (PSII) derived from Thermosynecoccus vulcanus as representative membrane proteins, we evaluated whether NPDGC12KK could solubilize membran...

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.

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.

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.

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.

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.

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.

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]

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.

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin–biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAP), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.

Photosynthetic membrane proteins convert solar light into chemical energy in a significantly high efficiency. Up-to-date reports of the photosynthetic bacterium suggest that such effective light conversion is due to the energy transfer between two light-harvesting (LH) protein complexes that are patterned in two dimensions. In this report, LH complex isolated from Rb. sphaeroides was immobilized onto a patterned gold surface with self-assembled monolayers (SAMs) and lipid bilayers at two main objectives: (1) micron-scale patterning of LH complex, and (2) prevention of quenching for pattern observation.

In a bacterial photosynthesis, 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. Here, we present a novel strategy to assemble both LH2 and LH1-RC into a solid-supported lipid bilayer. Spectroscopic and microscopic analyses revealed that both LH2 and LH1-RC were incorporated into the solid-supported lipid bilayer without denaturation. Through a stepwise-assembling procedure, LH2 and LH1-RC were separately organized in the lipid-domain-structured area.