Hidetaka Akita

Publications (89)411.52 Total impact

• Article: Gene Silencing via RNAi and siRNA Quantification in Tumor Tissue Using MEND, a Liposomal siRNA Delivery System.

  Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima
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  ABSTRACT: 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.Molecular Therapy (2013); doi:10.1038/mt.2013.57.
  Molecular Therapy 04/2013; · 6.87 Impact Factor
• Article: A Neutral Envelope-Type Nanoparticle Containing pH-Responsive and SS-Cleavable Lipid-Like Material as a Carrier for Plasmid DNA.

  Hidetaka Akita, Ryohei Ishiba, Hiroto Hatakeyama, Hiroki Tanaka, Yusuke Sato, Kota Tange, Masaya Arai, Kazuhiro Kubo, Hideyoshi Harashima
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  ABSTRACT: 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).
  Advanced healthcare materials. 02/2013;
• Article: Therapeutic assessment of cytochrome c for the prevention of obesity through endothelial cell-targeted nanoparticulate system

  Md. Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo and Hideyoshi Harashima
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  ABSTRACT: Because the functional apoptosis-initiating protein, cytochrome C (CytC) is rapidly cleared from the circulation (t1/2 (half-life): 4 minutes), it cannot be used for in vivo therapy. We report herein on a hitherto unreported strategy for delivering exogenous CytC as a potential and safe antiobesity drug for preventing diet-induced obesity, the most common type of obesity in humans. The functional activity of CytC encapsulated in prohibitin (a white fat vessel-specific receptor)-targeted nanoparticles (PTNP) was evaluated quantitatively, as evidenced by the observations that CytC-loaded PTNP causes apoptosis in primary adipose endothelial cells in a dose-dependent manner, whereas CytC alone did not. The delivery of a single dose of CytC through PTNP into the circulation disrupted the vascular structure by the targeted apoptosis of adipose endothelial cells in vivo. Intravenous treatment of CytC-loaded PTNP resulted in a substantial reduction in obesity in high-fat diet (HFD) fed wild-type (wt) mice, as evidenced by the dose-dependent prevention of the percentage of increase in body weight and decrease in serum leptin levels. In addition, no detectable hepatotoxicity was found to be associated with this prevention. Thus, the finding highlights the promising potential of CytC for use as an antiobesity drug, when delivered through a nanosystem.
  Molecular therapy. 01/2013;
• Article: Therapeutic Assessment of Cytochrome C for the Prevention of Obesity Through Endothelial Cell-targeted Nanoparticulate System.

  Md Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Taichi Ishitsuka, Hideyoshi Harashima
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  ABSTRACT: Because the functional apoptosis-initiating protein, cytochrome C (CytC) is rapidly cleared from the circulation (t(1/2) (half-life): 4 minutes), it cannot be used for in vivo therapy. We report herein on a hitherto unreported strategy for delivering exogenous CytC as a potential and safe antiobesity drug for preventing diet-induced obesity, the most common type of obesity in humans. The functional activity of CytC encapsulated in prohibitin (a white fat vessel-specific receptor)-targeted nanoparticles (PTNP) was evaluated quantitatively, as evidenced by the observations that CytC-loaded PTNP causes apoptosis in primary adipose endothelial cells in a dose-dependent manner, whereas CytC alone did not. The delivery of a single dose of CytC through PTNP into the circulation disrupted the vascular structure by the targeted apoptosis of adipose endothelial cells in vivo. Intravenous treatment of CytC-loaded PTNP resulted in a substantial reduction in obesity in high-fat diet (HFD) fed wild-type (wt) mice, as evidenced by the dose-dependent prevention of the percentage of increase in body weight and decrease in serum leptin levels. In addition, no detectable hepatotoxicity was found to be associated with this prevention. Thus, the finding highlights the promising potential of CytC for use as an antiobesity drug, when delivered through a nanosystem.Molecular Therapy (2013); doi:10.1038/mt.2012.256.
  Molecular Therapy 01/2013; · 6.87 Impact Factor
• Article: Particle Tracking Analysis for the Intracellular Trafficking of Nanoparticles Modified with African Swine Fever Virus Protein p54-derived Peptide.

  Hidetaka Akita, Kaoru Enoto, Hiroki Tanaka, Hideyoshi Harashima
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  ABSTRACT: Previous studies showed that the cytoplasmic transport of nanoparticles to the nucleus is driven by a vesicular sorting system. Artificial approaches for targeting a microtubule-associating motor complex is also a challenge. We describe herein the development of a liposomal nanoparticle, the surface of which is modified with stearylated octa-arginine (STR-R8), and a dynein light chain (LC8)-associated peptide derived from an African swine fever virus protein p54 (p54(149-161)) with polyethyleneglycol (PEG) as a spacer (p54(149-161)-PEG/R8-liposomal nanoparticles (LNPs)). The p54(149-161)-PEG/R8-LNPs preferentially gain access to the nucleus, resulting in a one- to two-order of magnitude higher transfection activity in comparison with p54(149-161)-free nanoparticles (PEG/R8-LNPs). Further studies of particle tracking in HeLa cells stably expressing green fluorescent protein (GFP)-tagged tubulin (GFP/Tub-HeLa) indicate that p54(149-161) stimulated the transport of nanoparticles along fibrous tubulin structures. Moreover, a part of the p54(149-161)-PEG/R8-LNPs appeared to undergo quasi-straight transport without sharing the tracks corresponding to PKH67, the plasma membrane of which had been prestained with a marker just before transfection, while corresponding movement was never observed in the case of PEG/R8-LNPs. These findings suggest that a portion of the p54(149-161)-modified nanoparticles can use microtubule-dependent transport without the need for an assist by a vesicular sorting system.Molecular Therapy (2012); doi:10.1038/mt.2012.235.
  Molecular Therapy 11/2012; · 6.87 Impact Factor
• Article: RNA Interference-Based Silencing Reveals the Regulatory Role of Fatty Acid-Binding Protein 4 in the Production of IL-6 and Vascular Endothelial Growth Factor in 3T3-L1 Adipocytes.

  Kazuaki Kajimoto, Shiharu Takayanagi, Shun Sasaki, Hidetaka Akita, Hideyoshi Harashima
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  ABSTRACT: The fatty acid-binding protein 4 (FABP4) is believed to play an important role in maintaining glucose and lipid homeostasis. However, the physiological functions of FABP4 in adipocytes have not been fully elucidated because of difficulties associated with the effective transfection of small interfering RNA (siRNA) to differentiated adipocytes. The aim of this study was to clarify the physiological roles of FABP4 in adipocytes by establishing an efficient, universal technique for endogenous gene silencing in fully differentiated 3T3-L1 cells. Confocal-based three-dimensional observations demonstrated that, in traditionally cultured 3T3-L1 cells, multilayers of undifferentiated cells were formed. As a result, small interfering RNA failed to reach many of the differentiated cells. To solve this problem, we developed a reliable method, denoted as density-based separation followed by replating of enriched adipocytes in a monolayer (DREAM) and, using the developed method, succeeded in a significant knockdown of FABP4. Loss-of-function analyses revealed that FABP4 regulates the expression of IL-6 and vascular endothelial growth factor (VEGF) mediated by the protease-activated receptor 1 (PAR1), a thrombin receptor, in adipocytes. In addition, the basal IL-6 production was partially suppressed by PAR1 knockdown. Moreover, we also demonstrated that IL-6 stimulates the proliferation of primary endothelial cells isolated from murine adipose tissue. These findings indicate that FABP4 may have a crucial role in modulating IL-6 and vascular endothelial growth factor as angiogenesis inducers stimulated by the cellular action of thrombin on adipocytes via PAR1. These findings promise to be helpful for developing an understanding of physiological counterparts with respect to the inflammatory and angiogenic properties of adipose tissue.
  Endocrinology 09/2012; 153(11):5629-36. · 4.46 Impact Factor
• Article: Pretreatment of Hepatocyte Growth Factor Gene Transfer Mediated by Octaarginine Peptide-Modified Nanoparticles Ameliorates LPS/D-Galactosamine-Induced Hepatitis.

  Yasuhiro Hayashi, Ryoichi Mizuno, Khalil A Ikramy, Hidetaka Akita, Hideyoshi Harashima
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  ABSTRACT: We previously reported that an octaarginine- and pH-sensitive fusogenic peptide-modified multifunctional envelope-type nano device (R8-GALA-MEND) produces a high level of gene expression in the liver. In this study, we report on an examination of whether this gene delivery system exerts potent hepatoprotective effects against lipopolysaccharide/D-galactosamine (LPS/D-GalN)-induced acute liver injury. In vivo-jetPEI(™)-Gal, a commercially available in vivo transfection reagent, was used as a reference. The systemic administration of the R8-GALA-MEND or in vivo-jetPEI(™)-Gal showed that the latter was more toxic than the R8-GALA-MEND, indicating that R8-GALA-MEND is a safer system than in vivo-jetPEI(™)-Gal. Pretreatment with R8-GALA-MEND or in vivo-jetPEI(™)-Gal loaded with hepatocyte growth factor (HGF) pDNA inhibited serum GPT and GOT levels from becoming elevated. However, the survival rate of the mice was significantly enhanced in the case of R8-GALA-MEND, but not for the in vivo-jetPEI(™)-Gal treatment. These results demonstrate that R8-GALA-MEND has the potential for use in the pretreatment of an acute liver injury.
  Nucleic acid therapeutics. 09/2012; 22(5):360-3.
• Article: Vascular-targeted nanotherapy for obesity: Unexpected passive targeting mechanism to obese fat for the enhancement of active drug delivery.

  Md Nazir Hossen, Kazuaki Kajimoto, Hidetaka Akita, Mamoru Hyodo, Hideyoshi Harashima
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  ABSTRACT: We previously reported that nanoparticles (NPs) modified with a prohibitin-homing peptide ligand via a short PEG(2kDa)-spacer could deliver its pay-load into the cytoplasm of endothelial cells in murine adipose tissue and escape from endosomes/lysosomes in vitro. We herein report, for the first time, on a dual-targeting strategy for mediating the enhanced targeting activity of NPs to adipose endothelial cells in diet-induced obesity (DIO). The targeted accumulation of prohibitin-targeted nanoparticles (PTNP), modified with a peptide ligand via a long PEG-linker, was significantly increased in white fat vessels of normal healthy mice compared to the other non-PEGylated targeted NPs, whereas the undesired accumulation of PTNP in the liver was considerably reduced. These results demonstrate that the PEGylation of targeted NPs is a critical factor in maximizing the in vivo targeted delivery of NPs and can be attributed to a significant decrease in recognition by the reticuloendothelial system. After systemic administration to DIO mice, PTNP exclusively accumulated in both adipose vessels and angiogenic clusters of obese fat cells. Surprisingly, PEGylated NPs with no active targeting moieties also accumulated in these clusters, demonstrating that the nanoscaled carriers passively accumulate in clusters via a mechanism similar to that for the enhanced permeability and retention effect, as has been well established in tumor targeting. Therefore, the enhanced delivery of PTNP appears to be mediated by both passive accumulation to angiogenic regions and active recognition by endothelial cells. Thus, the systemic administration of a proapoptotic peptide with the delivery via PTNP significantly reduced the body weight of DIO mice, as evidenced by the targeted ablation of adipose endothelial cells. These findings are potentially useful in terms of the design and development of vascular-targeted nanotherapy in the effective control of obesity.
  Journal of Controlled Release 09/2012; 163(2):101-10. · 5.73 Impact Factor
• Article: FRET from quantum dots to photodecompose undesired acceptors and report the condensation and decondensation of plasmid DNA.

  Vasudevanpillai Biju, Abdulaziz Anas, Hidetaka Akita, Edakkattuparambil Sidharthan Shibu, Tamitake Itoh, Hideyoshi Harashima, Mitsuru Ishikawa
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  ABSTRACT: Protection of genes against enzymatic degradation and overcoming of cellular barriers are critical for efficient gene delivery. The effectiveness of gene delivery by nonviral vectors depends mostly on the extent of DNA packaging or condensation. We show that Förster resonance energy transfer (FRET)-mediated photodecomposition of undesired acceptors in doubly labeled plasmid DNA (pDNA) and FRET recovery after acceptor photodecomposition (FRET-RAP) are effective methods for the detection of DNA condensation and decondensation. Our hypothesis is that undesired acceptors within the Förster distance of highly-photostable donors in precondensed DNA can be selectively photodecomposed by FRET. We investigate this hypothesis by the random labeling of pcDNA3.1-GL3 and pUC18DNA with quantum dots (QDs) as the energy donor and AlexaFluor594 or Cy5 as the acceptor. At first, the random labeling generates efficient FRET, also called intrinsic FRET, in precondensed DNA, which prevents us from decoding any changes in the FRET efficiency during DNA condensation. Next, we suppressed the intrinsic FRET by the FRET-mediated photodecomposition of acceptors within the Förster distance of QDs. Conversely, many acceptors kept intact beyond the Förster distance provide us with high FRET efficiency during the condensation of pDNA using protamine. Further, the FRET efficiency is significantly decreased during the decondensation of DNA using heparan sulfate and glutathione. The random labeling of DNA using excess acceptors around photostable donors followed by the FRET-mediated photodecomposition of undesired acceptors can be a promising method for not only the sensitive detection of DNA condensation by FRET but also the customization of biomolecular sensors.
  ACS Nano 04/2012; 6(5):3776-88. · 10.77 Impact Factor
• Article: Effect of the anchor in polyethylene glycol-lipids on the transfection activity of PEGylated cationic liposomes encapsulating DNA.

  Kenji Kusumoto, Hidetaka Akita, Ayman El-Sayed, Hideyoshi Harashima
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  ABSTRACT: 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.
  Biological & Pharmaceutical Bulletin 01/2012; 35(4):445-8. · 1.66 Impact Factor
• Article: Development of an Efficient Short Interference RNA (siRNA) Delivery System with a New pH-Sensitive Cationic Lipid.

  Yusuke Sato, Hiroto Hatakeyama, Mamoru Hyodo, Hidetaka Akita, Hideyoshi Harashima
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  ABSTRACT: 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.
  YAKUGAKU ZASSHI 01/2012; 132(12):1355-63. · 0.37 Impact Factor
• Article: Multifunctional envelope-type nano device (MEND) for organelle targeting via a stepwise membrane fusion process.

  Yuma Yamada, Hidetaka Akita, Hideyoshi Harashima
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  ABSTRACT: 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.
  Methods in enzymology 01/2012; 509:301-26. · 1.90 Impact Factor
• Article: Efficient intracellular delivery of nucleic acid pharmaceuticals using cell-penetrating peptides.

  Ikuhiko Nakase, Hidetaka Akita, Kentaro Kogure, Astrid Gräslund, Ulo Langel, Hideyoshi Harashima, Shiroh Futaki
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  ABSTRACT: Over the last 20 years, researchers have designed or discovered peptides that can permeate membranes and deliver exogenous molecules inside a cell. These peptides, known as cell-penetrating peptides (CPPs), typically consist of 6-30 residues, including HIV TAT peptide, penetratin, oligoarginine, transportan, and TP10. Through chemical conjugation or noncovalent complex formation, these structures successfully deliver bioactive and membrane-impermeable molecules into cells. CPPs have also gained attention as an attractive vehicle for the delivery of nucleic acid pharmaceuticals (NAPs), including genes/plasmids, short oligonucleotides, and small interference RNAs and their analogues, due to their high internalization efficacy, low cytotoxicity, and flexible structural design. In this Account, we survey the potential of CPPs for the design and optimization of NAP delivery systems. First, we describe the impact of the N-terminal stearylation of CPPs. Endocytic pathways make a major contribution to the cellular uptake of NAPs. Stearylation at the N-terminus of CPPs with stearyl-octaarginine (R8), stearyl-(RxR)(4), and stearyl-TP10 prompts the formation of a self-assembled core-shell nanoparticle with NAPs, a compact structure that promotes cellular uptake. Researchers have designed modifications such as the addition of trifluoromethylquinoline moieties to lysine residues to destabilize endosomes, as exemplified by PepFect 6, and these changes further improve biological responsiveness. Alternatively, stearylation also allows implantation of CPPs onto the surface of liposomes. This feature facilitates "programmed packaging" to establish multifunctional envelope-type nanodevices (MEND). The R8-MEND showed high transfection efficiency comparable to that of adenovirus in non-dividing cells. Understanding the cellular uptake mechanisms of CPPs will further improve CPP-mediated NAP delivery. The cellular uptake of CPPs and their NAP complex involves various types of endocytosis. Macropinocytosis, a mechanism which is also activated in response to stimuli such as growth factors or viruses, is a primary pathway for arginine-rich CPPs because high cationic charge density promotes this endocytic pathway. The use of larger endosomes (known as macropinosomes) rather than clathrin- or caveolae-mediated endocytosis has been reported in macropinocytosis which would also facilitate the endocytosis of NAP nanoparticles into cells.
  Accounts of Chemical Research 12/2011; 45(7):1132-9. · 21.64 Impact Factor
• Article: Stearylated INF7 peptide enhances endosomal escape and gene expression of PEGylated nanoparticles both in vitro and in vivo.

  Ayman El-Sayed, Tomoya Masuda, Hidetaka Akita, Hideyoshi Harashima
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  ABSTRACT: We previously reported on a stearylated INF7 peptide (str-INF7), which enhances the endosomal escape of an octaarginine (R8)-modified liposomal particle encapsulating plasmid DNA (pDNA) in a fusion-independent manner. This study examined whether this peptide derivative enhanced the endosomal escape and gene expression of PEGylated liposomes encapsulating pDNA. We used a PEGylated, R8-modified multifunctional envelope-type nanodevice (R8-MEND) as a model for PEGylated liposomes. Polyethylene glycol 2000 (PEG2000) attached to two different anchors, distearoylphosphatidylethanolamine (DSPE-PEG) or dimyristoylphosphatidylethanolamine (DMPE-PEG), was used to modify the R8-MEND in the presence or absence of two different concentrations of str-INF7. Modification of the PEGylated R8-MEND with str-INF7 resulted in luciferase gene expression levels in HeLa cells that were 73-fold and 24-fold higher than the corresponding value for an unmodified MEND in the case of DSPE-PEG and DMPE-PEG, respectively. The endosomal escape of the PEGylated R8-MEND was improved by str-INF7, as confirmed by confocal laser scanning microscopy. Furthermore, modification with str-INF7 enhanced the hepatic gene expression of the R8-MEND modified with DSPE-PEG and DMPE-PEG by 95-fold and 1885-fold, respectively, after intravenous injection in mice. Collectively, these data demonstrate that str-INF7 can be a useful device for enhancing the endosomal escape even for PEGylated liposomes encapsulating pDNA.
  Journal of Pharmaceutical Sciences 11/2011; 101(2):879-82. · 3.06 Impact Factor
• Article: Endosomal escape and the knockdown efficiency of liposomal-siRNA by the fusogenic peptide shGALA.

  Yu Sakurai, Hiroto Hatakeyama, Yusuke Sato, Hidetaka Akita, Kentaro Takayama, Sachiko Kobayashi, Shiroh Futaki, Hideyoshi Harashima
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  ABSTRACT: An siRNA that specifically silences the expression of mRNA is a potential therapeutic agent for dealing with many diseases including cancer. However, the poor cellular uptake and bioavailability of siRNA remains a major obstacle to clinical development. For efficient delivery to tumor tissue, the pharmacokinetics and intracellular trafficking of siRNA must be rigorously controlled. To address this issue, we developed a liposomal siRNA carrier, a multi-functional nano device (MEND). We describe herein an approach for systemic siRNA delivery to tumors by combining the MEND system with shGALA, a fusogenic peptide. In cultured cell experiments, shGALA-modification enhanced the endosomal escape of siRNA encapsulated in a polyethylene glycol modified MEND (PEG-MEND), resulting in an 82% knockdown of the target gene. In vivo systemic administration clarified that the shGALA-modified MEND (shGALA-MEND) showed 58% gene silencing in tumor tissues at a dose of 4 mg of siRNA/kg body weight. In addition, a significant inhibition of tumor growth was observed only for the shGALA-MEND and no somatic or hepatic toxicity was observed. Given the above data, this peptide-modified delivery system, a shGALA-MEND has great potential for the systemic delivery of therapeutic siRNA aimed at cancer therapy.
  Biomaterials 08/2011; 32(24):5733-42. · 7.40 Impact Factor
• Article: Cell penetrating peptide-mediated systemic siRNA delivery to the liver.

  Yasuhiro Hayashi, Jun Yamauchi, Ikramy A Khalil, Kazuaki Kajimoto, Hidetaka Akita, Hideyoshi Harashima
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  ABSTRACT: The cell-penetrating peptide (CPP) is one of the most attractive tools for efficiently delivering biomolecules to a target organelle. Here, we describe the use of octaarginine (R8)-modified lipid nanoparticles for the efficient and targeted in vivo delivery of siRNA to the liver. In this study, SR-BI (a scavenger receptor class B, member 1) was targeted by this nanoparticle. Our results demonstrate that R8-modified lipid nanoparticles can be used for the efficient and targeted delivery of liver siRNA to induce the specific knock-down of an endogenous gene with minimum liver toxicity and immune response, and that this CPP based technology holds considerable promise for further in vivo biological applications of siRNA.
  International journal of pharmaceutics 07/2011; 419(1-2):308-13. · 2.96 Impact Factor
• Article: KALA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine.

  Sharif M Shaheen, Hidetaka Akita, Takashi Nakamura, Shota Takayama, Shiroh Futaki, Atsushi Yamashita, Ryo Katoono, Nobuhiko Yui, Hideyoshi Harashima
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  ABSTRACT: DNA vaccines are a new-generation vaccines that elicit an immunological response against a wide-variety of antigens with frequent mutations. However, an effective non-viral vector for genetically engineered DNA to dendritic cells is yet to be developed. We previously reported that an octaarginine (R8)-modified tetra-lamellar multi-functional envelope-type nano device (R8-T-MEND) increases transfection efficiency in dendritic cell cultures (JAWS II). The critical structural elements of the R8-T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes, and two endosome-fusogenic outer envelopes. While the gene expression was drastically enhanced by R8-T-MEND, antigen presentation using an epitope-encoding plasmid DNA remains an obstacle for future non-viral vectors in DNA vaccinations. In the present study, we upgraded the function of R8-T-MEND by improving the membrane-fusion processes with endosome- and nuclear membranes by incorporating the KALA peptide, and by reducing the charge ratio (+/-), in an attempt to accelerate intra-nuclear decondensation. The resulting KALA-modified T-MEND (R8/KALA-T-MEND) showed an approximately 20-fold higher transgene expression compared with the conventional R8-T-MEND in JAWS II, and exceeded that of Lipofectamine PLUS, a commercially available transfection reagent. Furthermore, significant antigen presentation of a specific epitope (SIINFEKL) was observed for the R8/KALA-T-MEND but was not detected for the conventional T-MEND or Lipofectamine PLUS when an ovalbumin (OVA)-encoding plasmid DNA was transfected. It thus appears that the R8/KALA-T-MEND has the potential for use as a vector in DNA vaccinations.
  Biomaterials 06/2011; 32(26):6342-50. · 7.40 Impact Factor
• Article: Improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of GALA and maltotriose.

  Hidetaka Akita, Tomoya Masuda, Takashi Nishio, Kenichi Niikura, Kuniharu Ijiro, Hideyoshi Harashima
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  ABSTRACT: The successful control of intracellular trafficking (i.e., endosomal escape and nuclear delivery) is prerequisite for the development of a gene delivery system. In the present study, we developed an in vivo hepatic gene delivery system using a plasmid DNA (pDNA)-encapsulating lipid envelope-type nanoparticle, to which we refer as a multifunctional envelope-type nanodevice (MEND). The critical structural elements of the MEND are a DNA/protamine condensed core coated with lipid bilayers including serum-resistant cationic lipids. Intravenous administration of bare MEND represents minimal transfection activity. For the surface modification of functional devices, hydrophobic moieties were chemically attached, which are shed in the spontaneous orientation outward from the MEND surface by anchoring to the lipid bilayers. Modification of the pH-dependent fusogenic peptide GALA as an endosome escape induced transfection activity by 1 and 2 orders of magnitude. In an attempt to induce the nuclear delivery of pDNA, maltotriose, a recently characterized nuclear localization signal, was additionally modified. As a result, transfection activity further enhanced by 1 order of magnitude, and it reached to the higher level obtained for a conventional lipoplex and an in vivo jetPEI-Gal, with less hepatic toxicity. The data show that the combination of GALA and maltotriose results in a highly potent functional device that shows an enhanced endosomal escape and nuclear delivery in vivo.
  Molecular Pharmaceutics 06/2011; 8(4):1436-42. · 4.78 Impact Factor
• Article: Dual imaging of mRNA and protein production: an investigation of the mechanism of heterogeneity in cationic lipid-mediated transgene expression.

  Hidetaka Akita, Yuko Umetsu, Dai Kurihara, Hideyoshi Harashima
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  ABSTRACT: 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.
  International journal of pharmaceutics 05/2011; 415(1-2):218-20. · 2.96 Impact Factor
• Article: Functional improvement of an IRQ-PEG-MEND for delivering genes to the lung.

  Taichi Ishitsuka, Hidetaka Akita, Hideyoshi Harashima
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  ABSTRACT: The targeted delivery of genes to endothelial cells is a potential strategy for curing certain types of disorders including cancer, inflammation and obesity. We previously reported that a liposome (IRQ-LP) modified with the IRQ peptide (IRQRRRR) was taken up by cells via a unique pathway, namely caveolar endocytosis, a cellular uptake pathway that is involved in the blood-to-tissue uptake of macromolecules in vascular endothelial cells. In the present study, we initally investigated the effect of IRQ peptide-modification on the biodistribution of poly(ethyleneglycol) (PEG)-coated liposomes (PEG-LP) after i.v. administration. The IRQ peptide-modified PEG-LP (IRQ-PEG-LP), as well as the PEG-LP were found to be mainly accumulated in the liver. Nevertheless, the fold increase in the lung accumulation of IRQ-PEG-LP, compared to the PEG-LP (approximately 20-folds) was substantially higher than other tissues (<5-fold). Thus, IRQ could function as a target ligand for lungs. We then used the IRQ peptide as a model for a ligand for targeting normal tissue endothelial cells, and then applied it to a gene delivery system. We previously developed a multifunctional envelope-type nano device (MEND), in which plasmid DNA is condensed using a polycation to form a core particle that is encapsulated in a lipid envelope. We modified the IRQ-modified PEG to the MEND (IRQ-PEG-MEND) and marker gene expression was evaluated after i.v. administration. However the transgene expression of the IRQ-PEG-MEND in lungs was low. This is most likely due to the inhibitory effect of the PEG spacer on intracellular trafficking (especially endosomal escape) of the IRQ-PEG-MEND. To overcome the dilemma associated with PEGylation, we improved the MEND system from the point of view of PEG length, lipid chain of the PEG derivative, the polycation and cationic lipid. As a result, transgene expression in lungs was enhanced in stepwise manner, and was finally improved by 5 orders of magnitude compared with the original IRQ-PEG-MEND. Overcoming the dilemma of PEGylation is critical issue for in vivo applications of gene delivery targeting endothelial cells.
  Journal of Controlled Release 05/2011; 154(1):77-83. · 5.73 Impact Factor