The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices

Faint electric treatment-induced rapid and efficient delivery of extraneous hydrophilic molecules into the cytoplasm

Effective delivery of extraneous molecules into the cytoplasm of the target cells is important for several drug therapies. Previously, we showed effective in vivo transdermal delivery of naked siRNA into skin cells induced by faint electric treatment (ET) iontophoresis, and significant suppression of target mRNA levels (Kigasawa et al., Int. J. Pharm., 2010). This result indicates that electricity promoted the delivery of siRNA into cytoplasm. In the present study, we analyzed the intracellular delivery of naked anti-luciferase siRNA by faint ET, and found that the luciferase activity of cells expressing luciferase was reduced by in vitro ET like in vivo iontophoresis. Cellular uptake of fluorescent-label siRNA was increased by ET, while low temperature exposure, macropinocytosis inhibitor amiloride and caveolae-mediated endocytosis inhibitor filipin significantly prevented siRNA uptake. These results indicate that the cellular uptake mechanism involved endocytosis. In addition, voltage sensitive fluorescent dye DiBAC4 (3) penetration was increased by ET, and the transient receptor potential channel inhibitor SKF96365 reduced siRNA uptake, suggesting that faint ET reduced membrane potentials by changing intracellular ion levels. Moreover, to analyze cytoplasmic delivery, we used in-stem molecular beacon (ISMB), which fluoresces upon binding to target mRNA in the cytoplasm. Surprisingly, cytoplasmic ISMB fluorescence appeared rapidly and homogeneously after ET, indicating that cytoplasmic delivery is markedly enhanced by ET. In conclusion, we demonstrated for the first time that faint ET can enhance cellular uptake and cytoplasmic delivery of extraneous molecules

Effect of Phosphatidylserine and Cholesterol on Membrane-mediated Fibril Formation by the N-terminal Amyloidogenic Fragment of Apolipoprotein A-I

Here, we examined the effects of phosphatidylserine (PS) and cholesterol on the fibril-forming properties of the N-terminal 1‒83 fragment of an amyloidogenic G26R variant of apoA-I bound to small unilamellar vesicles. A thioflavin T fluorescence assay together with microscopic observations showed that PS significantly retards the nucleation step in fibril formation by apoA-I 1‒83/G26R, whereas cholesterol slightly enhances fibril formation. Circular dichroism analyses demonstrated that PS facilitates a structural transition from random coil to α-helix in apoA-I 1‒83/G26R with great stabilization of the α-helical structure upon lipid binding. Isothermal titration calorimetry measurements revealed that PS induces a marked increase in capacity for binding of apoA-I 1‒83/G26R to the membrane surface, perhaps due to electrostatic interactions of positively charged amino acids in apoA-I with PS. Such effects of PS to enhance lipid interactions and inhibit fibril formation of apoA-I were also observed for the amyloidogenic region-containing apoA-I 8‒33/G26R peptide. Fluorescence measurements using environment-sensitive probes indicated that PS induces a more solvent-exposed, membrane-bound conformation in the amyloidogenic region of apoA-I without affecting membrane fluidity. Since cell membranes have highly heterogeneous lipid compositions, our findings may provide a molecular basis for the preferential deposition of apoA-I amyloid fibrils in tissues and organs

Immunochemical Approach for Monitoring of Structural Transition of ApoA-I upon HDL Formation Using Novel Monoclonal Antibodies

Apolipoprotein A-I (apoA-I) undergoes a large conformational reorganization during remodeling of high-density lipoprotein (HDL) particles. To detect structural transition of apoA-I upon HDL formation, we developed novel monoclonal antibodies (mAbs). Splenocytes from BALB/c mice immunized with a recombinant human apoA-I, with or without conjugation with keyhole limpet hemocyanin, were fused with P3/NS1/1-Ag4-1 myeloma cells. After the HAT-selection and cloning, we established nine hybridoma clones secreting anti-apoA-I mAbs in which four mAbs recognize epitopes on the N-terminal half of apoA-I while the other five mAbs recognize the central region. ELISA and bio-layer interferometry measurements demonstrated that mAbs whose epitopes are within residues 1–43 or 44–65 obviously discriminate discoidal and spherical reconstituted HDL particles despite their great reactivities to lipid-free apoA-I and plasma HDL, suggesting the possibility of these mAbs to detect structural transition of apoA-I on HDL. Importantly, a helix-disrupting mutation of W50R into residues 44–65 restored the immunoreactivity of mAbs whose epitope being within residues 44–65 against reconstituted HDL particles, indicating that these mAbs specifically recognize the epitope region in a random coil state. These results encourage us to develop mAbs targeting epitopes in the N-terminal residues of apoA-I as useful probes for monitoring formation and remodeling of HDL particles

LMDP cleavage activity of γ-secretase assessed by HPLC analysis.

<p>LMDP cleavage activity of γ-secretase assessed by HPLC analysis.</p

Development of nanoparticles incorporating a novel liposomal membrane destabilization peptide (LMDP) for efficient drug release into cancer cells.

<p>This image depicts the concept of the carriers incorporating LMDP into the liposomal membrane (LMDP-lipo). The lipid bilayer membrane of LMDP-lipo is destabilized by cleaving LMDP as a trigger for membrane fusion. The destabilization promotes cargos release into the cell.</p

Time lapse imaging of LMDP-lipo by CLSM.

<p>(a) DiI-labeled and calcein-encapsulated LMDP-lipo (EPC/DOPE/CHEMS/5 mol% LMDP) was added to A549 cells and incubated at 37°C. CM-DiI (red) and calcein (green) were observed at intervals of 30 sec by CLSM. Asterisks indicate the same LMDP-lipo. Scale bars, 2 µm. (b) Schematic image of fluorescence measurement. Fluorescence intensities of CM-DiI and calcein were measured at 0, 2 and 4 µm from the centers of dots indicating fluorescent-labeled liposomes taken up by the cells. Changes in fluorescence intensity in 3 dots/cell for 5 cells at the indicated times were analyzed for CM-DiI (c) and calcein (d) using NIS-Elements software.</p

Development of Nanoparticles Incorporating a Novel Liposomal Membrane Destabilization Peptide for Efficient Release of Cargos into Cancer Cells

<div><p>In anti-cancer therapy mediated by a nanoparticle-based drug delivery system (DDS), overall efficacy depends on the release efficiency of cargos from the nanoparticles in the cancer cells as well as the specificity of delivery to tumor tissue. However, conventional liposome-based DDS have no mechanism for specifically releasing the encapsulated cargos inside the cancer cells. To overcome this barrier, we developed nanoparticles containing a novel liposomal membrane destabilization peptide (LMDP) that can destabilize membranes by cleavage with intramembranous proteases on/in cancer cells. Calcein encapsulated in liposomes modified with LMDP (LMDP-lipo) was effectively released in the presence of a membrane fraction containing an LMDP-cleavable protease. The release was inhibited by a protease inhibitor, suggesting that LMDP-lipo could effectively release its cargo into cells in response to a cancer-specific protease. Moreover, when LMDP-lipo contained fusogenic lipids, the release of cargo was accelerated, suggesting that the fusion of LMDP-lipo with cellular membranes was the initial step in the intracellular delivery. Time-lapse microscopic observations showed that the release of cargo from LMDP-lipo occurred immediately after association of LMDP-lipo with target cells. Consequently, LMDP-lipo could be a useful nanoparticle capable of effective release of cargos specifically into targeted cancer cells.</p></div