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A standard treatment for primary peritoneal carcinoma has not been established to date. We describe a case in which this cancer was successfully treated by use of paclitaxel and carboplatin chemotherapy.An 80-year-old woman who presented with abdominal distension and right upper abdominal pain was diagnosed with massive ascites and an omentum tumor via abdominal computed tomography and magnetic resonance imaging (MRI); her ovaries were normal-sized. Serum levels of the tumor marker CA125 were above normal (170 U/mL), and aspiration cytology showed the presence of adenocarcinoma cells. Despite several examinations, the primary tumor was not detected. The patient underwent exploratory laparotomy and was diagnosed with primary peritoneal carcinoma. She received combination chemotherapy consisting of paclitaxel and carboplatin. Serum CA125 levels returned to normal, and an MRI showed no evidence ofa tumor.Paclitaxel and carboplatin combination chemotherapy is effective for treatment of primary peritoneal adenocarcinomas.

Efficient DNA carriers are needed as a gene medication for curing brain disorders. In the present study, the function of a neutral lipid envelope-type nanoparticle (LNP) encapsulating pDNA was evaluated after intracerebroventricular administration. The lipid envelope was composed of a series of SS-cleavable and pH-activated lipid like materials (ssPalm) including myristic acid, vitamin A and vitamin E in the hydrophobic scaffold (LNPssPalmM, LNPssPalmA, LNPssPalmE, respectively). The LNPssPalmA and LNPssPalmE were extensively distributed in the corpus callosum, and then gene expression occurred mainly astrocytes in this region, while not in LNPssPalmM. The recombinant human ApoE3-dependent enhancement of the uptake into an astrocyte-derived cell line (KT-5) was observed in LNPssPalmA and LNPssPalmE. Thus, ApoE in the brain plays a key role in the cellular uptake of these particles by astrocytes, and this uptake is dependent on the structure of the hydrophobic scaffold.

For successful cancer gene therapy via intravenous (i.v.) administration, it is essential to optimize the stability of carriers in the systemic circulation and the cellular association after the accumulation of the carrier in tumor tissue. However, a dilemma exists regarding the use of poly(ethylene glycol) (PEG), which is useful for conferring stability in the systemic circulation, but is undesirable for the cellular uptake and the following processes. We report the development of a PEG-peptide-lipid ternary conjugate (PEG-Peptide-DOPE conjugate (PPD)). In this strategy, the PEG is removed from the carriers via cleavage by a matrix metalloproteinase (MMP), which is specifically expressed in tumor tissues. An in vitro study revealed that the PPD-modified gene carrier (Multifunctional Envelope-type Nano Device: MEND) exhibited pDNA expression activity that was dependent on the MMP expression level in the host cells. In vivo studies further revealed that the PPD was potent in stabilizing MEND in the systemic circulation and facilitating tumor accumulation. Moreover, the i.v. administration of PPD or PEG/PPD dually-modified MEND resulted in the stimulation of pDNA expression in tumor tissue, as compared with a conventional PEG-modified MEND. Thus, MEND modified with PPD is a promising device, which has the potential to make in vivo cancer gene therapy achievable.

Vectorial transport of bile acids across hepatocytes is a major driving force for bile flow, and bile acid retention in the liver causes hepatotoxicity. The basolateral and apical transporters for bile acids are thought to be targets of drugs that induce cholestasis. Previously, we constructed polarized LLC-PK1 cells that express both a major bile acid uptake transporter human Na+/taurocholate cotransporting polypeptide (SLC10A1) (NTCP) and the bile acid efflux transporter human bile salt export pump (ABCB 11) (BSEP) and showed that monolayers of such cells can be used to characterize vectorial transcellular transport of bile acids. In the present study, we investigated whether cholestasis-inducing drugs could inhibit bile acid transport in such cells. Because fluorescent substrates allow the development of a high-throughput screening method, we examined the transport by NTCP and BSEP of fluorescent bile acids as well as taurocholate. The aminofluorescein-tagged bile acids, chenodeoxycholylglycylamidofluorescein and cholylglycylamidofluorescein, were substrates of both NTCP and BSEP, and their basal-to-apical transport rates across coexpressing cell monolayers were 4.3 to 4.5 times those of the vector control, although smaller than for taurocholate. The well known cholestatic drugs, rifampicin, rifamycin SV, glibenclamide, and cyclosporin A, reduced the basal-to-apical transport and the apical efflux clearance of taurocholate across NTCP- and BSEP-coexpressing cell monolayers. Further analysis indicated that the drugs inhibited both NTCP and BSEP. Our study suggests that such coexpressing cells can provide a useful system for the identification of inhibitors of these two transport systems, including potential drug candidates.

Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 degrees C and 4 degrees C, was investigated in living cells. Compared with 37 degrees C, the uptake of R8-liposomes was only slightly reduced at 4 degrees C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 degrees C. When introduced at 4 degrees C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 degrees C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 degrees C, was less prominent compared with those introduced at 37 degrees C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA.

Na+-taurocholate-cotransporting peptide (NTCP)/SLC10A1 and bile salt export pump (BSEP)/ABCB11 synergistically play an important role in the transport of bile salts by the hepatocyte. In this study, we transfected human NTCP and BSEP or rat Ntcp and Bsep into LLC-PK1 cells, a cell line devoid of bile salts transporters. Transport by these cells was characterized with a focus on substrate specificity between rats and humans. The basal to apical flux of taurocholate across NTCP- and BSEP-expressing LLC-PK1 monolayers was 10 times higher than that in the opposite direction, whereas the flux across the monolayer of control and NTCP or BSEP single-expressing cells did not show any vectorial transport. The basal to apical flux of taurocholate was saturated with a Kmvalue of 20 μM. Vectorial transcellular transport was also observed for cholate, chenodeoxycholate, ursodeoxycholate, their taurine and glycine conjugates, and taurodeoxycholate and glycodeoxycholate, whereas no transport of lithocholate was detected. To evaluate the respective functions of NTCP and BSEP and to compare them with those of rat Ntcp and Bsep, we calculated the clearance by each transporter in this system. A good correlation in the clearance of the examined bile salts (cholate, chenodeoxycholate, ursodeoxycholate, and their taurine or glycine conjugates) was observed between transport by human and that of rat transporters in terms of their rank order: for NTCP, taurine conjugates > glycine conjugates > unconjugated bile salts, and for BSEP, unconjugated bile salts and glycine conjugates > taurine conjugates. In conclusion, the substrate specificity of human and rat NTCP and BSEP appear to be very similar at least for monovalent bile salts under physiological conditions.

A polypseudorotaxane consisting of a linear polyethylenimine with Mn of 22,000 (LPEI22k) and gamma-cyclodextrins (gamma-CDs; LPEI22k/gamma-CD) has been examined as a gene carrier. The polyplex formation with luciferase-encoding plasmid DNA (pDNA), intracellular trafficking of polyplex, cytotoxicity, and transfection efficiency were evaluated by various characteristic methods. LPEI22k/gamma-CD formed a pDNA polyplex at higher N/P ratios than LPEI22k; this suggests that the gamma-CD threading sterically interfered with the polyplex formation. In addition, the zeta potentials of the polyplex significantly decreased due to the reduction in charge density of LPEI22k caused by gamma-CD threading. The cellular uptake of pDNA in the LPEI22k/gamma-CD polyplex was enhanced by free gamma-CDs released from the polyplex that might accelerate the cellular uptake through enhanced membrane affinity. LPEI22k/gamma-CD significantly increased cell viability even at high N/P ratios, and the polyplex showed high transfection efficacy. The low cytotoxicity and high gene expression of LPEI22k/gamma-CD are advantageous to polyplex administration in vivo.

Background The efficient nuclear delivery of plasmid DNA (pDNA) is essential for the development of a promising non-viral gene vector. In an attempt to achieve nuclear delivery, NLS-mu, a novel pDNA condenser, was prepared. This consists of mu, a highly potent polypeptide for condensing the pDNA, and a SV40 T antigen-derived nuclear localization signal (NLSSV40). Methods The utility of NLS-mu was assessed in terms of green fluorescent protein (GFP) expression after cytoplasmic and nuclear microinjection of GFP-encoding pDNA along with the transfection, and compared with mu and poly-L-lysine (PLL). Trans-gene expression after cytoplasmic microinjection was affected by the efficiencies of nuclear transfer and following intra-nuclear transcription. To evaluate the nuclear transfer process separately, we introduced a parameter, a nuclear transfer score (NT score), which was calculated as the trans-gene expression after cytoplasmic microinjection divided by that after nuclear microinjection. Results As expected, the rank order of trans-gene expression after the transfection and cytoplasmic microinjection was NLS-mu > mu > PLL. However, the calculated NT scores were unexpectedly ranked as mu = NLS-mu > PLL, suggesting that mu, and not NLSSV40, is responsible for the nuclear delivery of pDNA. In addition, confocal images of rhodamine-labeled pDNA indicated that pDNA condensed with mu and NLS-mu was delivered as a condensed form. In comparing the nuclear transcription, the rank order of trans-gene expression after nuclear microinjection was PLL = NLS-mu > mu, suggesting that intra-nuclear transcription is inhibited by efficient condensation by mu, and is avoided by the attachment of NLSSV40. Conclusions Collectively, NLS-mu, which consists of chimeric functions, is an excellent DNA condenser, and the process is based on mu-derived nuclear transfer and NLSSV40-derived efficient intra-nuclear transcription. Copyright © 2005 John Wiley & Sons, Ltd.

In this study, we developed novel double-membranous non-viral gene delivery system modified with SV-40 T antigen-derived nuclear localization signal (NLS-DMEND) for delivery of luciferase plasmid DNA to nucleus of non-dividing mouse bone marrow-derived dendritic cells (BMDC). Intracellular trafficking and gene expression of NLS-DMEND in the BMDC were evaluated. Condensed DNA was observed in the nucleus by confocal laser scanning microscopy, and the NLS-DMEND induced significant luciferase activity in the BMDC. It was suggested that the condensed DNA particle transferred into nucleus via energy dependent manner, since the nuclear transfer was inhibited by metabolic inhibitors. In conclusion, condensed plasmid DNA was delivered into the nucleus of non-dividing BMDC by NLS-DMEND.

An in vitro cell culture model that mimics in vivo extracellular environment would be useful in developing in vivo gene delivery system. In the present study, a parallel flow model was applied to investigate the impact of convective flow on cellular uptake and transfection activity in endothelial cells. LipofectAMINE PLUS and adenovirus were used as model vectors, which bind cells via electrostatic- and ligand-receptor interactions, respectively. Whereas a convective flow increased the total amount of vector passing through the flow chamber by 3 orders of magnitude, uptake was increased by less than 10-fold, suggesting that the flow severely inhibited cellular uptake by reducing the retention time in the chamber and/or by diminishing the affinity between the cell and vector. Moreover, the uptake of both vectors was increased in a shear stress-dependent manner to a comparable extent, suggesting that the effect of flow on the cellular uptake was not significant. In contrast, transfection efficiency (TE), expressed as the transfection activity normalized by the cellular uptake of vectors was dramatically stimulated by shear stress, only when LipofectAMINE PLUS was used. Since the activities of the CMV promoter were unaffected by a shear stress, it is possible that altered intracellular trafficking may responsible for the improvement in lipoplex-mediated TE, presumably related to the cellular uptake pathway.

Lactoferrin (Lf) is an iron-binding glycoprotein belonging to the transferrin (Tf) family. Lf was reported to cross the blood brain barrier (BBB) via receptor-mediated transcytosis in an in vitro model of the BBB. In the present study, we compared the in vivo brain uptake of Lf with that of OX26, an anti-Tf receptor antibody, and Tf. These three proteins were radiolabeled with 125I and administered to rats by i.v. injection. We found that Lf was more rapidly eliminated from the blood compared with OX26 and Tf (The half-life of Lf was approximately 8 and 6 times shorter than that of OX26 and Tf, respectively; the area under the blood concentration–time curve of Lf was approximately 15 and 17 times smaller than that of OX26 and Tf, respectively), and mainly accumulated in the liver, spleen, and kidney. Markedly high brain uptake was observed for Lf relative to Tf and OX26. Lf might be useful as a ligand for facilitating drug delivery into the brain.

A multifunctional envelope-type nano device (MEND) for use in the delivery of siRNA expression plasmids is described. The plasmid DNA encoding anti-luciferase short interfering RNA (siRNA) was condensed by poly-L-lysine (PLL) and packaged into the MEND. The silencing effect of the MEND(PLL) showed a 96% inhibition of luciferase activities in a co-transfection study. The silencing effect was maintained at more than 60%, even under the 100-fold diluted conditions. In the luciferase transformed cells, however, the MEND(PLL) showed no significant silencing effect (10%), indicating heterogeneity in transfection by the MEND(PLL). To solve this problem, the DNA condensing agents were optimized by comparing PLL, stearyl octaarginine (STR-R8) and protamine (Prot). No difference in silencing effect (95-97%) was found among these MENDs in a co-transfection study. However, the MEND(Prot) showed a 70% silencing effect in the transformed cells. These results suggest that the MEND(Prot) has less heterogeneity in transfection, while the MEND(PLL) and the MEND(STR-R8) have large heterogeneities. These results demonstrate that MEND(Prot) is a promising gene delivery system for siRNA expression plasmids with less heterogeneity associated with the transfection.

An efficient delivery system is required if antisense oligodeoxynucleotides (ODN) are to be utilized for gene therapy. We report herein on the development of a novel ODN delivery system, ODN-encapsulated nano particles (ODN-ENP) using an efficient and simple packaging method. The ODN-ENP consists of a condensed ODN particle and a lipid envelope, which can be equipped with various functional devices for the efficient delivery of ODN with a small diameter (150 nm). The encapsulation efficiency and ODN recovery of ODN-ENP were significantly higher than those of other packaging methods, such as a stabilized antisense-lipid particles method or a freeze-thaw method. Furthermore, the time required for the preparation of the ODN-ENP was shorter than the other methods. The method developed in this study is a simple and efficient packaging method for ODN with a condensed nano particle in lipid-envelope structure.

Progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by a mutation in the bile salt export pump (BSEP/ABCB11) gene. However, the mechanisms for the deficiency in the function of two mutations (E297G and D482G), which are frequently found in European patients, have not yet been identified. In the present study, we examined the transport activity and cellular localization of these two mutants in human embryonic kidney 293 and Madin-Darby canine kidney II cells, respectively. Introduction of E297G and D482G mutations into the human BSEP gene by site-directed mutagenesis resulted in a significant reduction in the BSEP expression level, which was associated with impaired membrane trafficking. Most of the D482G BSEP and some of the E297G BSEP underwent only core glycosylation and appeared to be predominantly located in the endoplasmic reticulum. The inhibition of proteasome function by MG132 resulted in the cellular accumulation of the core glycosylation form of the two mutants. In contrast, transport studies for taurocholate and glycocholate with membrane vesicles isolated from complementary DNA-transfected cells indicated that both mutations did not significantly affect the transport function of BSEP per se. In conclusion, E297G and D482G mutations result in impaired membrane trafficking, whereas the transport functions of these mutants remain largely unchanged.

We recently found that octaarginine modified liposomes (R8-Lip) can be efficiently internalized by cultured cells. The purpose of the present study was to quantitatively determine the effect of R8-density on the tissue distribution of R8-Lip in mice, using their clearance as an index. R8 was introduced in the form of stearylated R8 (STR-R8). The liposomes were composed of cholesterol and egg phosphatidylcholine and were labeled with [(3)H]cholesteryl hexadecyl ether. Various densities of R8 (3%, 10% and 30%) containing liposomes were prepared with a diameter of approximately 70-80 nm. The tissue distribution of R8-Lip was determined after their i.v. administration into mice and the effect of R8-density on tissue distribution was compared with uptake clearance, the calculated tissue distribution divided by the area under the blood concentration-time course. As results, R8-Lip were more rapidly eliminated from circulating blood and distributed to many tissues, especially liver depending on the R8-density. However, the tissue uptake clearance represented similar value to that of positively charge liposomes. Based on these results, we conclude that the R8-dependent increase in R8-Lip in various tissues tested indicates that positive charge, but not PTD function derived from R8 predominantly responsible for the enhancement of tissue distribution. Therefore, it is suggested that topology control of R8 is important to exhibit the PTD function.

Since endosomal escape and the nuclear delivery of plasmid DNA (pDNA) constitute major barriers for transgene expression, a quantitative evaluation of intracellular trafficking of pDNA would be highly desirable in terms of optimizing a nonviral gene delivery system. In the present study, a novel strategy is proposed for the quantification of rhodamine-labeled pDNA in endosomes/lysosomes, cytosol, and nucleus. Endosomes/lysosomes and nucleus were stained with LysoSensor DND-189 and Hoechst 33258, respectively, to distinguish them from the cytosol. The pixel areas of the clusters derived from the rhodamine were used as an index for the amount of pDNA. This approach was applied to the analysis of the intracellular trafficking of pDNA transfected by LipofectAMINE PLUS, stearylated octaarginine (STR-R8), and octaarginine (R8). In the case of R8, most of the pDNA was trapped by endosomes/lysosomes. STR-R8 exhibited endosomal escape followed by nuclear translocation in a time-dependent manner. LipofectAMINE PLUS was the most effective in rapidly delivering pDNA to the nucleus as well as the cytosol. These differences in the intracellular trafficking of pDNA correlated well with the transgene expression. Therefore, this method enables the quantitative analysis of the intracellular pharmacokinetics of pDNA and promises to provide useful information for optimizing nonviral gene delivery systems.

Inchin-ko-to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a "bile acid-independent" mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance-associated protein 2 (Mrp2; Abcc2)-mediated choleresis in vivo. Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)-dependent uptake of Mrp2-specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP-dependent uptake of estradiol 17-beta-D-glucuronide (E(2)17 beta G; by 265%), leukotriene C4 (LTC(4); by 161%), taurolithocholate-3-sulfate (TLC-3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2-deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin-treated liver tissue sections when compared with the vehicle-treated liver tissue sections. In conclusion, genipin may enhance the bile acid-independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases.

During gene therapy the concentration of plasmid DNA or oligonucleotides in the plasma can be quite different from their concentrations in the nucleus or cytosol where they exert their actions. For a better understanding of the apparent discrepancies between pharmacokinetics (PK) and pharmacodynamics (PD), a new concept for intracellular PK with an emphasis on the final efficacy of gene transcription is needed. Here, the conventional PK and intracellular PK and PD of non-viral gene delivery systems are discussed, together with a new concept, referred to as controlled intracellular disposition, which integrates these factors to gain a better understanding of gene expression in the nucleus. The importance of optimizing the system from a transcriptional point of view in the nucleus is also discussed. These new concepts must be integrated to develop an optimized non-viral gene delivery system.

Multidrugresistance-associatedprotein3(Mrp3/ABCC3),whichcanmediatethecellularextrusionofbileacids,isinducedonthehepaticsinusoidalmembraneofMrp2/ABCC2-deficientrats(Eisaihyperbilirubinemicrats;EHBRs)andphenobarbital-treatedSprague–Dawleyrats.Inthepresentstudy,thecorrelationbetweenthesinusoidaleffluxclearance(PS eff )of[ 3 H]taurocholate(TC)andthehepaticexpressionofMrp3wasinvestigatedusingperfusedliverfromtheserats.AsignificantcorrelationwasobservedbetweenthePS eff andthehepaticexpressionlevelofMrp3,suggestingacontributionbyMrp3tothesinusoidaleffluxofTC.Theresultsofthekineticanalysisalsosuggestedthatothertransporter(s)onthesinusoidalplasmamembranemayparticipateintheeffluxofTCunderphysiologicalconditions.ThecontributionofMrp3tothesinusoidaleffluxofTCinEHBRsandphenobarbital(80and40mg/kg)-treatedratswasrevealedtobe58%,48%,and31%,respectively. 2002ElsevierScience(USA).Allrightsreserved. Keywords:Mrp3;Bileacid;Perfusedliver;Sinusoidalefflux Theliverplaysanimportantroleinthehomeostasisofbileacidsinthebody.Thebileacidsynthesisisrate-limitedbythe7a-hydroxylationofcholesterolcatalyzedbycholesterol7a-hydroxylase(cytochromeP4507A;Cyp7A)[1].Alongwiththemetabolismand/orbio-synthesisofbileacids,thetransportersonthesinusoidalandcanalicularmembranessynergisticallyplayanim-portantroleinthehepaticdispositionofbileacids.IthasbeenestablishedthattheuptakeofbileacidsintohepatocytesismediatedbybothNa

Surgeons and pathologists encounter some problems in making accurate intraoperative rapid diagnoses. The processing of histological and cytological specimens is time consuming, and further more, only small regions can be examined. In this study, we evaluated the use of a contact microlaryngoscope in microlaryngeal surgery, in 5 cases of suspicious squamous epithelial atypia, such as dysplasia and squamous cell carcinoma. After surgery, the rapid findings (suspected diagnosis) obtained using contact microlaryngoscopy were compared with the histological specimens (final diagnosis). The diagnoses matched in all 5 cases. Thus, contact microlaryngoscopy is useful for intraoperative rapid diagnosis.

Purpose. Multidrug resistance-associated protein 3 (MRP3) was initially cloned as a hepatic transporter induced under cholestatic/hyperbilirubinemic conditions. In the present study, transport property of human MRP3 (hMRP3) was compared with that of rat MRP3 (rMRP3).

It is anticipated that nucleic acid medicines will be in widespread use in the future, since they have the potential to cure diseases based on molecular mechanisms at the level of gene expression. However, intelligent delivery systems are required to achieve nucleic acid therapy, since they can perform their function only when they reach the intracellular site of action. We have been developing a multifunctional envelope-type nanodevice abbreviated as MEND, which consists of functional nucleic acids as a core and lipid envelope, and can control not only biodistribution but also the intracellular trafficking of nucleic acids. In this chapter, we review the development and evolution of the MEND by providing several successful examples, including the R8-MEND, the KALA-MEND, the MITO-Porter, the YSK-MEND, and the PALM.

Mina Tamaru,* Hidetaka Akita,* Taichi Nakatani, Kazuaki Kajimoto, Yusuke Sato, Hiroto Hatakeyama, Hideyoshi Harashima Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan *These authors contributed equally to this work Abstract: An innovative drug delivery technology is urgently needed to satisfy unmet medical needs in treating various brain disorders. As a fundamental carrier for plasmid DNA or nucleic acids, we developed a liposomal nanoparticle (multifunctional envelope-type nano device [MEND]) containing a proton-ionizable amino lipid (YSK-MEND). Here we report on the impact of apolipoprotein E (ApoE) modification on the function of YSK-MEND in terms of targeting brain cells. The cellular uptake and function of YSK-MEND encapsulating short interference RNA or plasmid DNA were significantly improved as a result of ApoE modification in mouse neuron-derived cell lines (Neuro-2a and CAD). Intracerebroventricular administration of ApoE-modified YSK-MEND (ApoE/YSK-MEND) encapsulating plasmid DNA also resulted in higher transgene expression in comparison with YSK-MEND that was not modified with ApoE. Moreover, observation of fluorescence-labeled ApoE/YSK-MEND and expression of mCherry (fluorescence protein) derived from plasmid DNA indicated that this carrier might be useful for delivering and conferring transgene expression in neural stem cells and/or neural progenitor cells. Thus, this system may be a useful tool for the treatment of neurodegenerative disease. Keywords: brain, DNA, oligonucleotide, delivery, nanoparticles

SummaryHost innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5′-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5′-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.

We previously reported that a multifunctional envelope-type nano device (MEND) modified with a GALA peptide (GALA/MEND) exerted dual functions; effective targeting the pulmonary endothelium and endosomal escape. The GALA/MEND containing encapsulated siRNA was originally prepared by the film coated hydration method (GALA/MENDHyd). However, an ethanol dilution method was found to be more appropriate for scaling up the preparation of this liposomal nanoparticle. In this study, we report on the preparation of a GALA/MEND based on the principal of the ethanol dilution (GALA/MENDEto). The gene knockdown efficiency of the MENDHyd and MENDEt0H without GALA-modification was equivalent regardless of the method used in the preparation. The GALA/MENDEt0H induced more efficient gene silencing in the pulmonary endothelium (ED50; approximately 0.17 mg siRNA/kg) compared to the GALA/ MENDHyd. The GALA/MENDEt0H escaped from endosomes more rapidly than GALA/MENDHyd, while the pharmacokinetics and lung accumulation of GALA/MENDEtcni and GALA/MENDHyd were comparable after i.v. administration. Collectively, the ethanol dilution method improves the function of the GALA/MEND as a lung-targeting siRNA carrier. (C) 2014 Elsevier B.V. All rights reserved.

It is anticipated that nucleic acid medicines will be in widespread use in the future, since they have the potential to cure diseases based on molecular mechanisms at the level of gene expression. However, intelligent delivery systems are required to achieve nucleic acid therapy, since they can perform their function only when they reach the intracellular site of action. We have been developing a multifunctional envelope-type nanodevice abbreviated as MEND, which consists of functional nucleic acids as a core and lipid envelope, and can control not only biodistribution but also the intracellular trafficking of nucleic acids. In this chapter, we review the development and evolution of the MEND by providing several successful examples, including the R8-MEND, the KALA-MEND, the MITO-Porter, the YSK-MEND, and the PALM.

Biliary excretion of certain bile acids is mediated by multidrug resistance associated protein 2 (Mrp2) and the bile salt export pump (Bsep). In the present study, the transport properties of several bile acids were characterized in canalicular membrane vesicles (CMVs) isolated from Sprague--Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose Mrp2 function is hereditarily defective and in membrane vesicles isolated from Sf9 cells infected with recombinant baculovirus containing cDNAs encoding Mrp2 and Bsep. ATP-dependent uptake of [(3)H]taurochenodeoxycholate sulfate (TCDC-S) (K(m)=8.8 microM) and [(3)H]taurolithocholate sulfate (TLC-S) (K(m)=1.5 microM) was observed in CMVs from SD rats, but not from EHBR. In addition, ATP-dependent uptake of [(3)H]TLC-S (K(m)=3.9 microM) and [(3)H]taurocholate (TC) (K(m)=7.5 microM) was also observed in Mrp2- and Bsep-expressing Sf9 membrane vesicles, respectively. TCDC-S and TLC-S inhibited the ATP-dependent TC uptake into CMVs from SD rats with IC(50) values of 4.6 microM and 1.2 microM, respectively. In contrast, the corresponding values for Sf9 cells expressing Bsep were 59 and 62 microM, respectively, which were similar to those determined in CMVs from EHBR (68 and 33 microM, respectively). By co-expressing Mrp2 with Bsep in Sf9 cells, IC(50) values for membrane vesicles from these cells shifted to values comparable with those in CMVs from SD rats (4.6 and 1.2 microM). Moreover, in membrane vesicles where both Mrp2 and Bsep are co-expressed, preincubation with the sulfated bile acids potentiated their inhibitory effect on Bsep-mediated TC transport. These results can be accounted for by assuming that the sulfated bile acids trans-inhibit the Bsep-mediated transport of TC.

Purpose. It has been shown that plasma concentration and urinary excretion of bile acids is elevated under the cholestatic/hyperbilirubinemic conditions. Previously, it was demonstrated that the plasma concentration of bile acids was elevated in the multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The purpose of the present study was to compare the sinusoidal efflux clearance of taurocholate (TC) between Mrp2-deficient Eisai hyperbilirubinemic rats (EHBR) and normal rats.

We measured vapor-liquid equilibria (VLE) for the system of pentafluoroethane (HFC125)-difluoroethane (HFC152a) over the temperature range from 268.15 K to 373.15 K. Fitting VLE data obtained to an extended BWR equation of state, binary interaction parameters were correlated as a linear function of temperature. Applying the equation of state including the binary interaction parameters to refrigeration cycle, estimation of coefficient of performance (COP) revealed about 13.3 % energy-saving against chlorodifluoromethane (HCFC22), when the mole fraction of HFC125 is 0.44. As the condition of COP estimation, we assumed that temperature changes of out-side fluids were from 285.15 down to 280.15 K and 305.15 up to 310.15 K with 3 K in minimum temperature difference, respectively.

Charge-neutralized lipid envelope-type nanoparticles formed with SS-cleavable and pH-activated lipid-like materials (ssPalm) accumulate rapidly in the liver without forming aggregates in the blood circulation, and result in a liver-specific gene expression for a long duration (>2 weeks) with neither immunological responses nor hepatotoxicity after intraveneous administration, when it carries pDNA free from CpG-motifs.

A disorder in the brain endothelium is thought to be closely related to the pathophysiology of brain diseases. A method for delivering nucleic acids (i.e. short interference RNA; siRNA) to the brain endothelium should be an attractive strategy for curing brain disorders. A liposomal nanoparticle containing a proton-ionizable amino lipid was recently developed as a carrier of encapsulated siRNA. The aim of this study was to evaluate the utility of apolipoprotein E (ApoE) as a targeting ligand for mouse brain endothelial cells (MBEC4 cells). The cellular uptake of the ApoE-modified nanoparticles was gradually increased in an ApoE-density dependent manner. Furthermore, the ApoE-modified nanoparticles were taken up via both clathrin and caveolae mediated endocytosis, thus permitting them to avoid lysosomal degradation. Finally, endogenous gene silencing in MBEC4 cells was efficiently achieved depending on the ApoE-modification. Collectively, the ApoE-modified nanoparticle is a promising carrier for delivering nucleic acids to the brain endothelium.

Technologies for the transfection of antigen-encoding genes into the dendritic cells, and subsequent immune-activation are both prerequisites for a successful DNA vaccine. We herein report on the density-dependent enhancement of transgene expression by the simple modification by stearyl-conjugated KALA, an α-helical peptide (STR-KALA), onto a lipid envelope-type nanoparticle (the R8-MEND, an octaarginine-modified multifunctional envelope-type nano device). The enhanced transgene expression in the KALA-modified R8-MEND (R8/KALA-MEND) cannot be explained by cellular uptake and nuclear delivery efficacy. Thus, the post-nuclear delivery process (i.e. transcription), but not intracellular trafficking processes attributed the enhanced transfection efficacy. Microarray analyses revealed that transfection with the R8/KALA-MEND resulted in a greater perturbation in host genes expression in comparison with the R8-MEND and that this effect was time-dependent. Further pathway analyses in the category of transcription-related genes and a gene ontology analysis indicated that the R8/KALA-MEND stimulated the expression of transcription factors that are closely related to immune-activation (i.e. NF-kB and STAT). Inhibition of the transfection efficacy by blockage of the STAT pathways revealed that the enhanced transcription activity is the result of immune-stimulation. Collectively, the R8/KALA-MEND mounts a "switch-on" function that triggers signal transduction forward to the immune-stimulation analogous to an adjuvant, and consequently elicits active transcription.

Angiogenesis is one of crucial processes associated with tumor growth and development, and consequently a prime target for cancer therapy. Although tumor endothelial cells (TECs) play a key role in pathological angiogenesis, investigating phenotypical changes in neovessels when a gene expression in TEC is suppressed is a difficult task. Small interfering RNA (siRNA) represents a potential agent due to its ability to silence a gene of interest. We previously developed a system for in vivo siRNA delivery to cancer cells that involves a liposomal-delivery system, a MEND that contains a unique pH-sensitive cationic lipid, YSK05 (YSK-MEND). In the present study, we report on the development of a system that permits the delivery of siRNA to TECs by combining the YSK-MEND and a ligand that is specific to TECs. Cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) is a well-known ligand to alpha(V)beta(3) integrin, which is selectively expressed at high levels in TECs. We incorporated cRGD into the YSK-MEND (RGD-MEND) to achieve an efficient gene silencing in TECs. Quantitative RT-PCR and the 5' rapid amplification of cDNA ends PCR indicated that the intravenous injection of RGD-MEND at a dose of 4.0 mg/kg induced a significant RNAi-mediated gene reduction in TEC but not in endothelial cells of other organs. Finally, we evaluated the therapeutic potency of the RGD-MEND encapsulating siRNA against vascular endothelial growth factor receptor 2. A substantial delay in tumor growth was observed after three sequential RGD-MEND injections on alternate days. In conclusion, the RGD-MEND represents a new approach for the characterization of TECs and for us in anti-angiogenic therapy. (C) 2013 Elsevier B.V. All rights reserved.

Small interfering RNA (siRNA) would be predicted to function as a cancer drug, but an efficient siRNA delivery system is required for clinical development. To address this issue, we developed a liposomal siRNA carrier, a multifunctional envelope-type nanodevice (MEND). We previously reported that a MEND composed of a pH-sensitive cationic lipid, YSK05, showed significant knockdown in both in vitro and in tumor tissue by intratumoral injection. Here, we report on the development of an in vivo siRNA delivery system that is delivered by systemic injection and an analysis of the pharmacokinetics of an intravenously administered siRNA molecule in tumor tissue. Tumor delivery of siRNA was quantified by means of stem-loop primer quantitative reverse transcriptase PCR (qRT-PCR) method. PEGylation of the YSK-MEND results in the increase in the accumulation of siRNA in tumor tissue from 0.0079% ID/g tumor to 1.9% ID/g tumor. The Administration of the MEND (3 mg siRNA/kg body weight) showed about a 50% reduction in the target gene mRNA and protein. Moreover, we verified the induction of RNA interference by 5′ RACE-PCR method. The collective results reported here indicate that an siRNA carrier was developed that can deliver siRNA to a target cell in tumor tissue through an improved siRNA bioavailability.

Antiangiogenesis has been the focus of a new strategy for the treatment of obesity. However, little is known regarding the issue of whether targeting angiogenesis by nanoparticle-targeted therapeutic is advantageous or not in debugging the co-morbidity associated with diet-induced obesity (DIO) and the metabolic syndrome. We report herein on the positive effect of prohibitin (an adipose vascular marker)-targeted nanoparticle (PTNP) encapsulated in a proapoptotic peptide [(D)(KLAKLAK)(2), KLA] on DIO and dysfunctional adipose tissue, a major mediator of the metabolic syndrome, as evidenced by ectopic fat deposition. The systemic injection of DIO mice with a low dose of KLA-PTNP, rather than a bioconjugate composed of the same targeting peptide and KLA (Adipotide) resulted in a reduction in body weight, as evidenced by a significant decrease in serum leptin levels, in parallel with an antiobesity effect on dysfunctional adipose cells, including adipocytes and macrophages. In addition, the KLA-PTNP treatment resulted in a reduction in ectopic fat deposits in liver and muscle with the lipolytic action of elevated serum adiponectin, with no detectable hepatoxicity. Notably, drug delivery via PTNP that had accumulated in obese fat via the enhanced permeability and retention effect was enhanced by multivalent active targeting and cytoplasmic delivery into adipose endothelial cells via escaping from endosomes/lysosomes. Thus, vascular-targeted nanotherapy has the potential to contribute to the control of adipose function and ectopic fat deposition associated with obesity and the metabolic syndrome. (C) 2013 Elsevier B. V. All rights reserved.

The development of a carrier for the delivery of siRNA is a factor in the realization of RNA interference (RNAi) therapeutics. Modification of siRNA carriers with polyethylene glycol, i.e., PEGylation, is a general strategy for stabilizing a particle in the blood stream and delivering it to tissue or cells. However, it is well-known that, when a carrier is modified by PEGylation, it results in a significant inhibition of both cellular uptake and the endosomal escape process. In a previous study, we reported on the development of a multifunctional envelope-type nano device (MEND) for delivering siRNA and peptide-based functional devices for overcoming the effects conferred by PEGylation and succeeded in the delivery of siRNA to tumor tissue. In this study, we noticed that the pH-sensitive property, changing from neutral to cationic in response to a decrease in pH, could avoid the inhibition caused by PEGylation and succeeded in synthesizing a pH-sensitive cationic lipid, YSK05. The YSK05-MEND had a higher fusogenicity and potency for endosomal escape than other MENDs containing conventional cationic lipids. The PEGylated YSK05-MEND induced efficient gene silencing and avoided the inhibition of endosomal escape caused by PEGylation followed by optimization of the lipid composition. Furthermore, the intratumoral injection of the PEGylated YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids. Thus, the YSK05-MEND is a promising siRNA carrier for avoiding the inhibition in intracellular trafficking caused by PEGylation both in vitro and in vivo.

SS-cleavable proton-activated lipid-like material (ssPalm) functions as a key element in a lipid nanoparticle in which pDNA is encapsulated. The ssPalm contains dual sensing motifs that can respond to the intracellular environment; a proton-sponge unit (tertiary amines) that functions in response to an acidic environment (endosome/lysosome), and disulfide bonding that can be cleaved in a reducing environment (cytosol).

Modification of liposomal siRNA carriers with polyethylene glycol, i.e., PEGylation, is a generally accepted strategy for achieving in vivo stability and delivery to tumor tissue. However, PEGylation significantly inhibits both cellular uptake and the endosomal escape process of the carriers. In a previous study, we reported on the development of a multifunctional envelope-type nano device (MEND) for siRNA delivery and peptide-based functional devices for overcoming the limitations and succeeded in the efficient delivery of siRNA to tumors. In this study, we synthesized a pH-sensitive cationic lipid, YSK05, to overcome the limitations. The YSK05-MEND had a higher ability for endosomal escape than other MENDs containing conventional cationic lipids. The PEGylated YSK05-MEND induced efficient gene silencing and overcame the limitations followed by optimization of the lipid composition. Furthermore, the intratumoral administration of the YSK05-MEND resulted in a more efficient gene silencing compared with MENDs containing conventional cationic lipids. Collectively, these data confirm that YSK05 facilitates the endosomal escape of the MEND and thereby enhances the efficacy of siRNA delivery into cytosol and gene silencing.

A single cell contains a variety of organelles. Included among these organelles are the nucleus that regulates the central dogma, mitochondria that function as an energy plant, the Golgi apparatus that determines the destination of endogenous protein, and others. If it were possible to prepare a nano craft that could specifically target a specific organelle, this would open a new field of research directed toward therapy for various diseases. We recently developed a new concept of "Programmed Packaging," by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a nonviral gene-delivery system. Our attempts to target certain organelles (nucleus and mitochondria) are described here, mainly focusing on the construction of a tetra-lamellar MEND (T-MEND), and on methods for screening the organelle-specific fusogenic envelope. The critical structural elements of the T-MEND include an organelle-specific membrane-fusogenic inner envelope and a cellular membrane-fusogenic outer envelope. The resulting T-MEND can be utilized to overcome intracellular membrane barriers, since it involves stepwise membrane fusion. To deliver cargos into a target organelle in our strategy, the carriers must fuse with the organelle membrane. Therefore, we screened a series of lipid envelopes that have the potential for fusing with an organelle membrane by monitoring the inhibition of fluorescence resonance energy transfer and identified the optimal lipid conditions for nuclear and mitochondrial membrane fusion. Finally, we describe the delivery of a bioactive molecule targeted to the nucleus and mitochondria in living cells, demonstrating that this system can be useful for targeting various organelles.

The use of polyethylene glycol (PEG)-modified lipids (PEG-lipids) as a component of cationic liposomes impairs the cytoplasmic delivery of the encapsulated cargos by reducing endosomal escape. While this results in a loss of gene expression of encapsulated plasmid DNA, PEG-modification is useful in that it permits the formation of small, stabilized particles. In the present study, the dilemma associated with the use of PEG was overcome by modifying liposomes with stearylated INF7 (STR-INF7), a membrane fusion-independent destabilizer of endosomes, and substituting hydrophobic lipid-anchors in the PEG-lipid. The cationic liposomes modified with a series of PEG-lipids showed a drastically impaired transgene expression. However, the incorporation of STR-INF7 recovered the gene expression, and this was found to be mainly dependent on the type of PEG lipid-anchor used. Of note, the fold increase in transfection activity was highest in cholesterol-anchored PEG (>100-fold), whose enhanced endosomal escape was followed by imaging techniques. These data suggest that the structure of lipid-anchors in PEG affects the action of the peptides for inducing of endosomal escape.

In the 21st century, drug development has shifted toward larger molecules such as proteins and nucleic acids, which require the use of new chemical strategies. In this process, the drug delivery system plays a central role and intracellular targeting using nanotechnology has become a key technology for the development of successful new medicines. We have developed a new delivery system, a multifunctional envelope-type nanodevice (MEND) based on "Programmed Packaging." In this new concept of packaging, multifunctional nanodevices are integrated into a nanocarrier system according to a program designed to overcome all barriers during the course of biodistribution and intracellular trafficking. In this Account, we introduce our method for delivering nucleic acids or proteins to intracellular sites of action such as the cytosol, nucleus, and mitochondria and for targeting selective tissues in vivo via systemic administration of the nanodevices. First, we introduce an octaarginine-modified MEND (R8-MEND) as an efficient intracellular delivery system, designed especially for vaccinations and transgene expression. Many types of cells can internalize the R8-MEND, mainly by inducing macropinocytosis, and the MEND escapes from macropinosomes via membrane fusion, which leads to efficient antigen presentation via the major histocompatibility complex I pathway in antigen-presenting cells. In addition, the transfection activities of the R8-MEND in dividing cells, such as HeLa or A549 cells, are as high as those for adenovirus. However, because the R8-MEND cannot induce sufficient transgene activity in primary cultured dendritic cells, which are critical regulators of the immune response, we converted the R8-MEND into a tetralamellar MEND (T-MEND). The T-MEND uses a new packaging method and delivers condensed pDNA into the nucleus via fusion between the envelopes and the nuclear membrane. To achieve efficient transfection activity, we also optimized the decondensation of nucleic acids within the nucleus. To optimize mitochondrial drug delivery, we introduced the MITOPorter. Many types of materials can be packaged into this liposome-based nanocarrier and then delivered to mitochondria via membrane fusion mechanisms. Finally, we describe an integrated strategy for in vivo tumor delivery and optimization of intracellular trafficking. Successful tumor delivery typically requires coating the surfaces of nanoparticles with PEG, but PEG can also limit uptake by the reticuloendothelial system and reduce the efficiency of intracellular trafficking. Here we integrate the optimum biodistribution and intracellular trafficking of the MEND with an innovative strategy such as enzymatically cleavable PEG and a short membrane peptide, GALA. Some of these strategies will soon be tested in the clinic.

A single cell contains a variety of organelles. Included among these organelles are the nucleus that regulates the central dogma, mitochondria that function as an energy plant, the Golgi apparatus that determines the destination of endogenous protein, and others. If it were possible to prepare a nano craft that could specifically target a specific organelle, this would open a new field of research directed toward therapy for various diseases. We recently developed a new concept of "Programmed Packaging," by which we succeeded in creating a multifunctional envelope-type nano device (MEND) as a nonviral gene-delivery system. Our attempts to target certain organelles (nucleus and mitochondria) are described here, mainly focusing on the construction of a tetra-lamellar MEND (T-MEND), and on methods for screening the organelle-specific fusogenic envelope. The critical structural elements of the T-MEND include an organelle-specific membrane-fusogenic inner envelope and a cellular membrane-fusogenic outer envelope. The resulting T-MEND can be utilized to overcome intracellular membrane barriers, since it involves stepwise membrane fusion. To deliver cargos into a target organelle in our strategy, the carriers must fuse with the organelle membrane. Therefore, we screened a series of lipid envelopes that have the potential for fusing with an organelle membrane by monitoring the inhibition of fluorescence resonance energy transfer and identified the optimal lipid conditions for nuclear and mitochondrial membrane fusion. Finally, we describe the delivery of a bioactive molecule targeted to the nucleus and mitochondria in living cells, demonstrating that this system can be useful for targeting various organelles.

Heterogeneity of transgene expression is a severe disadvantage in the use of cationic lipid-mediated gene vectors. We previously demonstrated that heterogeneity of the post-nuclear delivery process, as well as intracellular trafficking (i.e. nuclear delivery) is a major determinant in the overall heterogeneity in gene expression, when plasmid DNA (pDNA) is transfected to HeLa cells using a lipoplex. In this study, we explored the mechanism underlying this heterogeneity in a post-nuclear transport process by the dual imaging of mRNA and its encoded protein (histone H2B-tagged mTFP1; mTFP1-H2B) in a single cell. To establish a highly sensitive imaging system for mRNA, we used fluorescence in situ hybridization (FISH) combined with tyramide signal amplification (TSA) and a semiconductor quantum dot (QD) probe. The mRNA expression and protein production were quantified by counting the total pixel intensity in the region of interest (r.o.i.) surrounding single cells. As a result, the correlation was poor in a scattered plot of mRNA expression versus protein production in individual cells. These findings demonstrate that cell-to-cell differences in the translation process are also a key factor in heterogeneous gene expression.