Publications by authors named "Tadanori Mayumi"

80 Publications

Amorphous nanosilicas induce consumptive coagulopathy after systemic exposure.

Nanotechnology 2012 Feb 4;23(4):045101. Epub 2012 Jan 4.

Laboratory of Toxicology and Safety Science, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan.

We previously reported that well-dispersed amorphous nanosilicas with particle size 70 nm (nSP70) penetrate skin and produce systemic exposure after topical application. These findings underscore the need to examine biological effects after systemic exposure to nanosilicas. The present study was designed to examine the biological effects. BALB/c mice were intravenously injected with amorphous nanosilicas of sizes 70, 100, 300, 1000 nm and then assessed for survival, blood biochemistry, and coagulation. As a result, injection of nSP70 caused fatal toxicity, liver damage, and platelet depletion, suggesting that nSP70 caused consumptive coagulopathy. Additionally, nSP70 exerts procoagulant activity in vitro associated with an increase in specific surface area, which increases as diameter reduces. In contrast, nSP70-mediated procoagulant activity was absent in factor XII-deficient plasma. Collectively, we revealed that interaction between nSP70 and intrinsic coagulation factors such as factor XII, were deeply related to nSP70-induced harmful effects. In other words, it is suggested that if interaction between nSP70 and coagulation factors can be suppressed, nSP70-induced harmful effects may be avoided. These results would provide useful information for ensuring the safety of nanomaterials (NMs) and open new frontiers in biological fields by the use of NMs.
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http://dx.doi.org/10.1088/0957-4484/23/4/045101DOI Listing
February 2012

Silica and titanium dioxide nanoparticles cause pregnancy complications in mice.

Nat Nanotechnol 2011 May 3;6(5):321-8. Epub 2011 Apr 3.

Department of Toxicology and Safety Science, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.

The increasing use of nanomaterials has raised concerns about their potential risks to human health. Recent studies have shown that nanoparticles can cross the placenta barrier in pregnant mice and cause neurotoxicity in their offspring, but a more detailed understanding of the effects of nanoparticles on pregnant animals remains elusive. Here, we show that silica and titanium dioxide nanoparticles with diameters of 70 nm and 35 nm, respectively, can cause pregnancy complications when injected intravenously into pregnant mice. The silica and titanium dioxide nanoparticles were found in the placenta, fetal liver and fetal brain. Mice treated with these nanoparticles had smaller uteri and smaller fetuses than untreated controls. Fullerene molecules and larger (300 and 1,000 nm) silica particles did not induce these complications. These detrimental effects are linked to structural and functional abnormalities in the placenta on the maternal side, and are abolished when the surfaces of the silica nanoparticles are modified with carboxyl and amine groups.
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http://dx.doi.org/10.1038/nnano.2011.41DOI Listing
May 2011

Systemic distribution, nuclear entry and cytotoxicity of amorphous nanosilica following topical application.

Biomaterials 2011 Apr 22;32(11):2713-24. Epub 2011 Jan 22.

Department of Toxicology and Safety Science, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

Currently, nanomaterials (NMs) with particle sizes below 100 nm have been successfully employed in various industrial applications in medicine, cosmetics and foods. On the other hand, NMs can also be problematic in terms of eliciting a toxicological effect by their small size. However, biological and/or cellular responses to NMs are often inconsistent and even contradictory. In addition, relationships among NMs physicochemical properties, absorbency, localization and biological responses are not yet well understood. In order to open new frontiers in medical, cosmetics and foods fields by the safer NMs, it is necessary to collect the information of the detailed properties of NMs and then, build the prediction system of NMs safety. The present study was designed to examine the skin penetration, cellular localization, and cytotoxic effects of the well-dispersed amorphous silica particles of diameters ranging from 70 nm to 1000 nm. Our results suggested that the well-dispersed amorphous nanosilica of particle size 70 nm (nSP70) penetrated the skin barrier and caused systemic exposure in mouse, and induced mutagenic activity in vitro. Our information indicated that further studies of relation between physicochemical properties and biological responses are needed for the development and the safer form of NMs.
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http://dx.doi.org/10.1016/j.biomaterials.2010.12.042DOI Listing
April 2011

Urban aerosols induce pro-inflammatory cytokine production in macrophages and cause airway inflammation in vivo.

Biol Pharm Bull 2010 ;33(5):780-3

Department of Toxicology and Safety Science, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

Urban air pollution, especially in developing countries, is a crucial environmental problem. Urban aerosols may contain various kinds of substances and induce harmful effects such as allergic diseases. Therefore, it is critical to clarify the biological effects of urban aerosols on human health. In this study, we evaluated the induction of airway inflammation in vitro and in vivo due to exposure of urban aerosols. We investigated cytokine production and nuclear factor-kappaB (NF-kappaB) activation after stimulation of macrophage cells by exposure of urban aerosols. Urban aerosols were found to induce the production of interleukin (IL)-8, tumor necrosis factor-alpha and IL-1beta on macrophage cells. In addition, we showed that NF-kappaB pathway regulated the urban aerosols-induced inflammatory cytokine response. Moreover, the intranasal administration of urban aerosols resulted in increases in the total cell number in bronchoalveolar lavage and infiltration of eosinophils in lung tissue. These results indicate that urban aerosols induce respiratory inflammation and onset of inflammatory disease due to an activation of the immune system.
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http://dx.doi.org/10.1248/bpb.33.780DOI Listing
December 2010

Carbon nanotubes elicit DNA damage and inflammatory response relative to their size and shape.

Inflammation 2010 Aug;33(4):276-80

Department of Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, Japan.

Carbon nanotubes (CNTs) have been one of the most extensively researched and developed nanomaterials. However, little concern has been placed on their safety. The biological effects of CNTs are believed to differ relative to size and shape. Thus, the relationship between the characteristics of CNTs and their safety needs to be evaluated. In this study, we examined the biological effects of different-sized multi-walled CNTs (MWCNTs) and single-walled CNTs (SWCNTs). Long and thick MWCNTs induced the strongest DNA damage while similar SWCNTs caused little effect. Comparison of inflammatory responses of various types of CNTs found that peritoneal CNT administration of long and thick MWCNTs increased the total cell number in abdominal lavage fluid in mice. These results indicate that long and thick MWCNT, but not short and thin MWCNT, cause DNA damage and severe inflammatory effects. These findings might provide useful information for constructing novel CNTs with safety.
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http://dx.doi.org/10.1007/s10753-010-9182-7DOI Listing
August 2010

The use of a mutant TNF-alpha as a vaccine adjuvant for the induction of mucosal immune responses.

Biomaterials 2009 Oct 30;30(29):5869-76. Epub 2009 Jul 30.

Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.

Safe and potent adjuvants are required in order to establish effective mucosal vaccines. Cytokines are promising adjuvants because they are human-derived safe biomaterial and display immune-modulating functions. We have created a mutant tumor necrosis factor-alpha (TNF-alpha), mTNF-K90R, that exhibits high bioactivity and resistance to proteases. Here, we examined the potential of mTNF-K90R as a mucosal adjuvant. Initially, we showed that intranasal co-administration of mTNF-K90R with ovalbumin (OVA) potently produced OVA-specific Immunoglobulin (Ig) G antibodies (Abs) in serum and IgA Abs both at local and distal mucosal sites compared to co-administration with wild-type TNF-alpha. The OVA-specific immune response was characterized by high levels of serum IgG1 and increased production of interleukin-4 (IL-4), IL-5 and IL-10 from splenocytes of immunized mice, suggesting a Th2 response. Furthermore, intranasal immunization with an antigen from influenza virus plus mTNF-K90R exhibited mucosal adjuvant activity for induction of both systemic and mucosal immune responses. Importantly, histopathological examination of the nasal tissue of mTNF-K90R treated mice detected no signs of toxicity. These findings suggest that mTNF-K90R is safe and effective mucosal adjuvant and this system may have potential application as a universal mucosal adjuvant system for mucosal vaccines improving the immune response to a variety of viral antigens.
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http://dx.doi.org/10.1016/j.biomaterials.2009.07.009DOI Listing
October 2009

TNF superfamily member, TL1A, is a potential mucosal vaccine adjuvant.

Biochem Biophys Res Commun 2009 Jul 4;384(3):296-300. Epub 2009 May 4.

Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation (NiBio), 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.

The identification of cytokine adjuvants capable of inducing an efficient mucosal immune response against viral pathogens has been long anticipated. Here, we attempted to identify the potential of tumor necrosis factor superfamily (TNFS) cytokines to function as mucosal vaccine adjuvants. Sixteen different TNFS cytokines were used to screen mucosal vaccine adjuvants, after which their immune responses were compared. Among the TNFS cytokines, intranasal immunization with OVA plus APRIL, TL1A, and TNF-alpha exhibited stronger immune response than those immunized with OVA alone. TL1A induced the strongest immune response and augmented OVA-specific IgG and IgA responses in serum and mucosal compartments, respectively. The OVA-specific immune response of TL1A was characterized by high levels of serum IgG1 and increased production of IL-4 and IL-5 from splenocytes of immunized mice, suggesting that TL1A might induce Th2-type responses. These findings indicate that TL1A has the most potential as a mucosal adjuvant among the TNFS cytokines.
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http://dx.doi.org/10.1016/j.bbrc.2009.04.115DOI Listing
July 2009

In vitro and in vivo tumor suppressive activity induced by human telomerase transcriptase-targeting antisense oligonucleotides mediated by cationic liposomes.

J Biosci Bioeng 2008 Sep;106(3):243-7

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

The objective of this study was to investigate the in vitro and in vivo influence of cationic liposomes on the tumor suppressive effect of antisense telomerase oligodeoxynucleotides to human cervical adenocarcinoma cells (HeLa). Antisense oligodeoxynucleotides (ASODN) against the human telomerase transcriptase (hTERT) served as telomerase inhibitors. The cholesterol derivative, 3beta [N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), was synthesized, and cationic liposomes (CL) were prepared using DC-Chol and dioleoylphosphatidylethanolamine (DOPE). The in vitro IC50 of the CL-ASODN complex was 1.88 mumol/l, while the IC50 of the cells treated with free ASODNs or CL alone was 25.24 mumol/l and 55.18 mumol/l, respectively. The CL-ASODN complex inhibited HeLa cell growth for at least 120 h. In vivo, the CL-ASODN complex inhibited the tumor growth rate by 55.11%, which increased to 89.47% when CL-ASODN was combined with 5-fluorouracil treatment. ASODNs alone failed to induce tumor-suppressive activity, suggesting that CL prepared from DOPE and DC-Chol can significantly enhance the growth inhibitory effect of ASODN on tumor cells both in vitro and in vivo.
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http://dx.doi.org/10.1263/jbb.106.243DOI Listing
September 2008

The therapeutic effect of TNFR1-selective antagonistic mutant TNF-alpha in murine hepatitis models.

Cytokine 2008 Nov 23;44(2):229-33. Epub 2008 Sep 23.

Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in a wide variety of inflammatory pathologies, such as hepatitis, via the TNF receptor-1 (TNFR1). To develop TNFR1-targeted anti-inflammatory drugs, we have already succeeded in creating a TNFR1-selective antagonistic mutant TNF-alpha (R1antTNF) and shown that R1antTNF efficiently inhibits TNF-alpha/TNFR1-mediated biological activity in vitro. In this study, we examined the therapeutic effect of R1antTNF in acute hepatitis using two independent experimental models, induced by carbon tetrachloride (CCl(4)) or concanavalin A (ConA). In a CCl(4)-induced model, treatment with R1antTNF significantly inhibited elevation in the serum level of ALT (alanine aminotransferase), a marker for liver damage. In a ConA-induced T-cell-mediated hepatitis model, R1antTNF also inhibited the production of serum immune activated markers such as IL-2 and IL-6. These R1antTNF-mediated therapeutic effects were as good as or better than those obtained using conventional anti-TNF-alpha antibody therapy. Our results suggest that R1antTNF may be a clinically useful TNF-alpha antagonist in hepatitis.
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http://dx.doi.org/10.1016/j.cyto.2008.07.003DOI Listing
November 2008

NK cells are migrated and indispensable in the anti-tumor activity induced by CCL27 gene therapy.

Cancer Immunol Immunother 2009 Feb 16;58(2):291-9. Epub 2008 Jul 16.

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou, Zhejiang, 310058, China.

Natural killer (NK) cells have been demonstrated could play an important role in the treatment of a number of tumors in mice. In the present study, chemokine CCL27, which be considered only selectively chemoattracts cutaneous lymphocyte antigen positive memory T cells and Langerhans cells, firstly demonstrated that it could induce the accumulation of NK cells into tumor by the intratumoral injection of CCL27-encoding fiber-mutant vector, AdRGD-CCL27. Experiments using spleen cell fractionation and RT-PCR showed CCL27 receptor, mCCR10, was strongly expressed in NK cells, suggesting the accumulation of NK cells in tumor was attributed to chemoattractant activity of CCL27 itself. Moreover, the combination of AdRGD-CCL27 and AdRGD-IL-12 induced the synergistic anti-tumor activity via NK-dependent manner and induced more NK cells infiltration into tumor nodule than that induced by AdRGD-CCL27 alone or AdRGD-IL-12 alone.
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http://dx.doi.org/10.1007/s00262-008-0554-xDOI Listing
February 2009

Combination of two fiber-mutant adenovirus vectors, one encoding the chemokine FKN and another encoding cytokine interleukin 12, elicits notably enhanced anti-tumor responses.

Cancer Immunol Immunother 2008 Nov 8;57(11):1657-64. Epub 2008 Mar 8.

Institute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, 388 Yuhangtang Road, 310058 Hangzhou, Zhejiang, People's Republic of China.

For achieving optimal cancer immunotherapy, it is anticipated that both the activation and infiltration of immune cells into tumor are indispensable. In the present study, fiber-mutant adenovirus vectors (Ad) encoding chemokine FKN, (AdRGD-FKN), and cytokine interleukin 12, (AdRGD-IL-12), were constructed. The in vivo gene expression of AdRGD was confirmed and the combination of both FKN and IL-12 encoding Ad elicited synergistic anti-tumor activity in ovarian carcinoma, which induced tumor regression in all tumor-bearing mice, while using FKN alone did not show notable tumor-suppressive effect. The treatment with both IL-12 and FKN induced long-term specific immunity against OV-HM tumors in tumor-rejected mice. The results of immunohistochemical staining for CD3+ and perforin-positive cells suggested that the failure of using FKN alone was because of the inactivation of infiltrated immune cells. In contrast, cotransduction with IL-12 and FKN could induce more activated tumor-infiltrating immune cells than that transducted with FKN or IL-12 alone. The results indicated that using both chemokine and cytokine might be a powerful tool and a promising way for effective cancer immunotherapy.
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http://dx.doi.org/10.1007/s00262-008-0499-0DOI Listing
November 2008

Creation and X-ray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor necrosis factor-alpha antagonist.

J Biol Chem 2008 Jan 14;283(2):998-1007. Epub 2007 Nov 14.

National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.

Tumor necrosis factor-alpha (TNF) induces inflammatory response predominantly through the TNF receptor-1 (TNFR1). Thus, blocking the binding of TNF to TNFR1 is an important strategy for the treatment of many inflammatory diseases, such as hepatitis and rheumatoid arthritis. In this study, we identified a TNFR1-selective antagonistic mutant TNF from a phage library displaying structural human TNF variants in which each one of the six amino acid residues at the receptor-binding site (amino acids at positions 84-89) was replaced with other amino acids. Consequently, a TNFR1-selective antagonistic mutant TNF (R1antTNF), containing mutations A84S, V85T, S86T, Y87H, Q88N, and T89Q, was isolated from the library. The R1antTNF did not activate TNFR1-mediated responses, although its affinity for the TNFR1 was almost similar to that of the human wild-type TNF (wtTNF). Additionally, the R1antTNF neutralized the TNFR1-mediated bioactivity of wtTNF without influencing its TNFR2-mediated bioactivity and inhibited hepatic injury in an experimental hepatitis model. To understand the mechanism underlying the antagonistic activity of R1antTNF, we analyzed this mutant using the surface plasmon resonance spectroscopy and x-ray crystallography. Kinetic association/dissociation parameters of the R1antTNF were higher than those of the wtTNF, indicating very fast bond dissociation. Furthermore, x-ray crystallographic analysis of R1antTNF suggested that the mutation Y87H changed the binding mode from the hydrophobic to the electrostatic interaction, which may be one of the reasons why R1antTNF behaved as an antagonist. Our studies demonstrate the feasibility of generating TNF receptor subtype-specific antagonist by extensive substitution of amino acids of the wild-type ligand protein.
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http://dx.doi.org/10.1074/jbc.M707933200DOI Listing
January 2008

Immune cell recruitment and cell-based system for cancer therapy.

Pharm Res 2008 Apr 22;25(4):752-68. Epub 2007 Sep 22.

College of Pharmaceutical Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, People's Republic of China.

Immune cells, such as cytotoxic T lymphocytes, natural killer cells, B cells, and dendritic cells, have a central role in cancer immunotherapy. Conventional studies of cancer immunotherapy have focused mainly on the search for an efficient means to prime/activate tumor-associated antigen-specific immunity. A systematic understanding of the molecular basis of the trafficking and biodistribution of immune cells, however, is important for the development of more efficacious cancer immunotherapies. It is well established that the basis and premise of immunotherapy is the accumulation of effective immune cells in tumor tissues. Therefore, it is crucial to control the distribution of immune cells to optimize cancer immunotherapy. Recent characterization of various chemokines and chemokine receptors in the immune system has increased our knowledge of the regulatory mechanisms of the immune response and tolerance based on immune cell localization. Here, we review the immune cell recruitment and cell-based systems that can potentially control the systemic pharmacokinetics of immune cells and, in particular, focus on cell migrating molecules, i.e., chemokines, and their receptors, and their use in cancer immunotherapy.
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http://dx.doi.org/10.1007/s11095-007-9443-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2279154PMC
April 2008

Effective tumor targeted gene transfer using PEGylated adenovirus vector via systemic administration.

J Control Release 2007 Sep 21;122(1):102-10. Epub 2007 Jun 21.

Department of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

Conjugation of polyethylene glycol to protein or particles (PEGylation) prolongs their plasma half-lives and promotes their accumulation in tumors due to enhanced permeability and retention (EPR) effect. Although PEGylation of adenovirus vectors (Ads) is an attractive strategy to improve the in vivo kinetics of conventional Ads, the EPR effect of PEGylated Ad (PEG-Ad) had not previously been reported. In this study, we prepared PEG-Ads with PEG at various modification ratios, injected them intravenously into tumor-bearing mice, and determined the blood kinetics, viral distribution, and gene expression patterns, respectively. In addition, we conducted a cancer therapeutic study of PEG-Ad encoding tumor necrosis factor (TNF)-alpha. The plasma half-life of PEG-Ad was longer than that of unmodified-Ad, and accumulation of PEG-Ad in tumor tissue increased as the PEG modification ratio increased. In particular, PEG-Ad with about 90% modification ratio showed higher (35 times) gene expression in tumor and lower (6%) in liver, compared with values for unmodified Ad. Moreover, PEG-Ad encoding TNF-alpha demonstrated not only stronger tumor-suppressive activity but also fewer hepatotoxic side effects compared with unmodified-Ad. PEGylation of Ad achieved tumor targeting through the EPR effect, and these attributes suggest that systemic injection of PEG-Ad has great potential as an anti-tumor treatment.
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http://dx.doi.org/10.1016/j.jconrel.2007.06.010DOI Listing
September 2007

Role of amino acid residue 90 in bioactivity and receptor binding capacity of tumor necrosis factor mutants.

Biochim Biophys Acta 2007 Aug 22;1774(8):1029-35. Epub 2007 May 22.

National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.

We have previously produced two bioactive lysine-deficient mutants of TNF-alpha (mutTNF-K90R,-K90P) and found that these mutants have bioactivity superior to wild-type TNF (wtTNF). Because these mutants contained same amino acid except for amino acid 90, it is unclear which amino acid residue is optimal for showing bioactivity. We speculated that this amino acid position was exchangeable, and this amino acid substitution enabled the creation of lysine-deficient mutants with enhanced bioactivity. Therefore, we produced mutTNF-K90R variants (mutTNF-R90X), in which R90 was replaced with other amino acids, to assay their bioactivities and investigated the importance of amino acid position 90. As a result, mutTNF-R90X that replaced R90 with lysine, arginine and proline were bioactive, while other mutants were not bioactive. Moreover, these three mutants showed bioactivity as good as or better than wtTNF. R90 replaced with lysine or arginine had especially superior binding affinities. These results suggest that the amino acid position 90 in TNF-alpha is important for TNF-alpha bioactivity and could be altered to improve its bioactivity to generate a "super-agonist".
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http://dx.doi.org/10.1016/j.bbapap.2007.05.003DOI Listing
August 2007

Creation of novel cell-penetrating peptides for intracellular drug delivery using systematic phage display technology originated from Tat transduction domain.

Biol Pharm Bull 2007 Feb;30(2):218-23

Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation, Osaka, Japan.

Many biologically active proteins need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm. Cell penetrating peptides (CPPs) have been developed to efficiently deliver a wide variety of cargo in a fully biological active form into a range of cell types for the treatment of multiple preclinical disease models. To further develop this methodology, we established a systematic approach to identify novel CPPs using phage display technology. Firstly, we screened a phage peptide library for peptides that bound to the cell membrane. Secondly, to assess functionality as intracellular carriers, we recombined cDNAs of binding peptides with protein synthesis inhibitory factor (PSIF) to create fusion proteins. Randomly chosen clones were cultured and expression of peptide-PSIF fusion proteins induced, followed by screening of protein synthesis activity in cells. Using this systematic approach, novel and effective CPPs were rapidly identified. We suggest that these novel cell-penetrating peptides can utilized as drug delivery tools for protein therapy or an analytical tool to study mechanisms of protein transduction into the cytoplasm.
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http://dx.doi.org/10.1248/bpb.30.218DOI Listing
February 2007

A strategy for efficient cross-presentation of CTL-epitope peptides leading to enhanced induction of in vivo tumor immunity.

J Control Release 2007 Jan 13;117(1):11-9. Epub 2006 Oct 13.

Department of Biotechnology and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

The activation of antitumor cytotoxic T-lymphocytes (CTLs) depends on how efficiently the relevant tumor antigen peptides are delivered into the major histocompatibility complex (MHC) class I presentation pathway in antigen presenting cells (APCs). An elegant approach to promote the peptide-MHC class I association has been described for enhanced peptide transportation into the endoplasmic reticulum (ER) by adding an ER insertion signal sequence (Eriss). Nevertheless, this approach does not appear potent enough to induce in vivo tumor protective immunity. Herein, we present a novel peptide-vaccine strategy based on the combined utilization of Eriss and fusogenic liposomes (FLs) capable of directly introducing encapsulated CTL-epitope peptides into the MHC class I pathway of APCs. APCs pulsed with free peptides, FL-encapsulated peptides, or FL-encapsulated Eriss-conjugated peptides exhibited comparable levels of antigen-presenting activity at early phases after pulsing. Interestingly, whereas in the first two methods the APC ability began to decline 40 to 60 h after pulsing, FL-encapsulated Eriss(+) peptides allowed APCs to retain peptide-presentation activity for at least 140 h. This advantage of FL-encapsulated Eriss(+) peptides correlated with the induction of more potent antitumor immunity compared with soluble Eriss(+) or Eriss(-) peptides or FL-encapsulated Eriss(-) peptides when they were administered in vivo. Thus, Eriss-conjugated CTL-epitope peptides encapsulated in FLs provide a highly efficient tumor-vaccine to enhance the induction of in vivo tumor immunity.
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http://dx.doi.org/10.1016/j.jconrel.2006.10.011DOI Listing
January 2007

A novel method for construction of gene fragment library to searching epitopes.

Biochem Biophys Res Commun 2006 Jul 24;346(1):198-204. Epub 2006 May 24.

Laboratory of Pharmaceutical Proteomics, National Institute of Biomedical Innovation, 7-6-8 Asagi, Saito, Ibaraki, Osaka 567-0085, Japan.

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.
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http://dx.doi.org/10.1016/j.bbrc.2006.05.103DOI Listing
July 2006

Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation.

J Biol Chem 2006 May 8;281(18):12673-81. Epub 2006 Mar 8.

Department of Pharmacology, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARgamma in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARgamma mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARgamma-deficient mouse brains, PPARgamma-RNA-silenced NSCs, and PPARgamma dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARgamma agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARgamma was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARgamma by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARgamma regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.
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http://dx.doi.org/10.1074/jbc.M513786200DOI Listing
May 2006

Bastadin 6, a spongean brominated tyrosine derivative, inhibits tumor angiogenesis by inducing selective apoptosis to endothelial cells.

Anticancer Drugs 2006 Mar;17(3):269-78

Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

Bastadin 6, a macrocyclic tetramer of a brominated tyrosine derivative, was isolated from a marine sponge and its anti-angiogenic activity was evaluated. Bastadin 6 was found to inhibit vascular endothelial growth factor (VEGF)- or basic fibroblast growth factor (bFGF)-dependent proliferation (IC50=0.052 micromol/l) of human umbilical vein endothelial cells (HUVECs) 20- to 100-fold selectively in comparison with normal fibroblast (3Y1) or several tumor cells (KB3-1, K562 and Neuro2A). Bastadin 6 also inhibited VEGF- or bFGF-induced tubular formation (0.1 micromol/l, 6 h treatment) and VEGF-induced migration (1 micromol/l, 4 h treatment) of HUVECs. Moreover, bastadin 6 almost completely blocked VEGF- or bFGF-induced in vivo neovascularization in the mice corneal assay and suppressed growth of s.c. inoculated A431 solid tumor in nude mice (100 mg/kg, i.p.). Bastadin 6 induced cell death of HUVECs with an apoptotic phenotype, whereas it showed no effect on the VEGF-induced auto-phosphorylation of VEGF receptors Flt-1 and KDR/Flk-1. These results suggest that the anti-angiogenic effect of bastadin 6 is closely related to selective induction activity of apoptosis against endothelial cells.
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http://dx.doi.org/10.1097/00001813-200603000-00005DOI Listing
March 2006

Non-methylated CpG motif packaged into fusogenic liposomes enhance antigen-specific immunity in mice.

Biol Pharm Bull 2006 Jan;29(1):105-9

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, Japan.

DNA rich in non-methylated CG motifs (CpGs) enhances induction of immune responses against co-administered antigen encoding genes. CpGs are therefore among the promising adjuvants known to date. However, naked plasmid DNA, even which contains CpG motifs, are taken up by antigen presenting cells via the endocytosis pathway. Endocytosed DNAs are thus degraded and their gene expression levels are inefficient. In this context, an effective plasmid delivery carrier is required for DNA vaccine development. We show in the present study that packaging plasmids containing CpGs into fusogenic liposomes (FL) derived from conventional liposomes and Sendai virus-derived active accessory proteins is an attractive method for enhancing the efficacy of a DNA vaccine. These CpG-enhanced plasmids (possessing 16 CpG repeats) that were packaged into FL, enhanced ovalbumin (OVA)-specific T cell proliferation and cytotoxic T cell activity after immunization. In fact, vaccination with CpG enhanced plasmid-loaded FL induced effective prophylactic effects compared with 13 repeats CpG containing plasmid in a tumor challenge experiment. Thus, the development of a CpG-enhanced DNA-FL genetic immunization system represents a promising tool for developing candidate vaccines against some of the more difficult infectious, parasitic, and oncologic disease targets.
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http://dx.doi.org/10.1248/bpb.29.105DOI Listing
January 2006

Vaccine efficacy of fusogenic liposomes containing tumor cell-lysate against murine B16BL6 melanoma.

Biol Pharm Bull 2006 Jan;29(1):100-4

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University.

Recent advances in tumor immunology have facilitated the development of cancer immunotherapy targeting tumor-associated antigens (TAAs). However, because TAAs were identified in only a few types of human cancer, novel vaccine strategies that utilize tumor cell-lysate (TCL), including unidentified TAAs as an antigen source, are needed. Herein, we describe the utility of fusogenic liposomes (FLs) as TCL-delivery carriers for both ex vivo dendritic cell-based vaccination and in vivo direct immunization in the murine B16BL6 melanoma model. As a result, both in vivo direct immunization and ex vivo immunization induced anti-B16 melanoma prophylactic effects. Ex vivo dendritic cell (DC)-mediated vaccination strategy exert more potent anti-tumor effect than direct immunization. Our results suggest that this flexible system is a promising approach for the development of versatile cancer immunotherapy regimes.
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http://dx.doi.org/10.1248/bpb.29.100DOI Listing
January 2006

Design and synthesis of a Tat-related gene transporter: a tool for carrying the adenovirus vector into cells.

Bioorg Med Chem Lett 2006 Feb 6;16(3):743-5. Epub 2005 Dec 6.

Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan.

A Tat-related peptide, acetyl-Gly-Arg-Arg-Arg-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, designed to transport an Adenovirus vector (Ad) into cells, was synthesized. The synthetic peptide was conjugated to Ad, which potentially can act as an efficient carrier of heterologous genes into cells. The Tat-related peptide was synthesized using the solid phase method and then was coupled to the heterofunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester. The resulting peptide-succinimidohexanoic acid N-hydroxysuccinimide ester was conjugated to Ad containing the luciferase gene. B16BL6 cells infected with the peptide-conjugated Ad luciferase gene construct exhibit a 50-fold greater luciferase activity than B16BL6 cells infected with wild-type Ad containing the luciferase gene.
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http://dx.doi.org/10.1016/j.bmcl.2005.08.116DOI Listing
February 2006

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release.

J Control Release 2005 Jul;105(3):344-53

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.
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http://dx.doi.org/10.1016/j.jconrel.2005.03.020DOI Listing
July 2005

Anti-tumor responses induced by chemokine CCL19 transfected into an ovarian carcinoma model via fiber-mutant adenovirus vector.

Biol Pharm Bull 2005 Jun;28(6):1066-70

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, Japan.

Considerable attention has recently been paid to the application of chemokines to cancer immunotherapy because of their chemotactic affinity for a variety of immune cells and because several chemokines are strongly angiostatic. In the present study, the recombinant adenovirus vectors encoding chemokine CCL19 or XCL1 in an E1 cassette (AdRGD-mCCL19 and AdRGD-mXCL1) were developed. The constructed fiber-mutant adenovirus vector, which contained the integrin-targeting Arg-Gly-Asp (RGD) sequence in the fiber knob, notably enhanced the transfection efficiency to OV-HM ovarian carcinoma cells compared to that induced by conventional adenovirus vector. The results of an in vitro chemotaxis assay for chemokine-encoding vector demonstrated that both AdRGD-mCCL19 and AdRGD-mXCL1 could induce the migration of cells expressing specific chemokine receptors. Of the two chemokine-encoding vectors evaluated in vivo, AdRGD-mCCL19 showed significant tumor-suppressive activity in B6C3F1 mice via transduction into OV-HM cells, whereas XCL1 did not exhibit any notable anti-tumor effects, suggesting that CCL19 may be a candidate for cancer immunotherapy.
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http://dx.doi.org/10.1248/bpb.28.1066DOI Listing
June 2005

RNA interference of PPARgamma using fiber-modified adenovirus vector efficiently suppresses preadipocyte-to-adipocyte differentiation in 3T3-L1 cells.

Gene 2005 Mar;348:157-65

Division of Cellular and Gene Therapy Products, National Institute of Health Sciences, Tokyo 158-8501, Japan.

The peroxisome proliferator-activated receptor (PPAR) gamma is regarded as a "master regulator" of adipocyte differentiation and is abundantly expressed in adipose. To understand the biological role of PPARgamma in adipose, RNA interference (RNAi) of PPARgamma should be a powerful tool. 3T3-L1 cell line serves an excellent model to investigate the mechanism of preadipocyte-to-adipocyte differentiation. However, this cell line is difficult to transfect by plasmid vectors and viral vectors. We optimized the transduction of both 3T3-L1 preadipocytes and adipocytes by means of fiber-modified adenovirus (Ad) vectors. Among the various vectors tested, polylysine modification of the C-terminal of the fiber knob most markedly improved the transduction efficiency in both 3T3-L1 preadipocytes and adipocytes. Then, we examined whether fiber-modified Ad vectors with polylysine peptides expressing the small interfering RNA (siRNA) for PPARgamma inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes. Oil red O staining and measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity indicated that the vectors effectively suppressed the differentiation of 3T3-L1 preadipocytes to adipocytes. These results suggested that the combination of fiber-modified Ad vectors containing polylysine peptides and RNAi is an effective tool for the study of the biological and physiological mechanism of adipogenesis in adiposity and diabetes using 3T3-L1 models. Ad vector-mediated RNAi for PPARgamma should also be useful to clarify the biological role of the PPARgamma pathway in various tissues in addition to adipose and for therapeutic application to a variety of diseases, including adiposity and diabetes.
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http://dx.doi.org/10.1016/j.gene.2005.01.005DOI Listing
March 2005

RNA interference of PPARgamma using fiber-modified adenovirus vector efficiently suppresses preadipocyte-to-adipocyte differentiation in 3T3-L1 cells.

Gene 2005 Mar;348:157-65

Division of Cellular and Gene Therapy Products, National Institute of Health Sciences, Tokyo 158-8501, Japan.

The peroxisome proliferator-activated receptor (PPAR) gamma is regarded as a "master regulator" of adipocyte differentiation and is abundantly expressed in adipose. To understand the biological role of PPARgamma in adipose, RNA interference (RNAi) of PPARgamma should be a powerful tool. 3T3-L1 cell line serves an excellent model to investigate the mechanism of preadipocyte-to-adipocyte differentiation. However, this cell line is difficult to transfect by plasmid vectors and viral vectors. We optimized the transduction of both 3T3-L1 preadipocytes and adipocytes by means of fiber-modified adenovirus (Ad) vectors. Among the various vectors tested, polylysine modification of the C-terminal of the fiber knob most markedly improved the transduction efficiency in both 3T3-L1 preadipocytes and adipocytes. Then, we examined whether fiber-modified Ad vectors with polylysine peptides expressing the small interfering RNA (siRNA) for PPARgamma inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes. Oil red O staining and measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity indicated that the vectors effectively suppressed the differentiation of 3T3-L1 preadipocytes to adipocytes. These results suggested that the combination of fiber-modified Ad vectors containing polylysine peptides and RNAi is an effective tool for the study of the biological and physiological mechanism of adipogenesis in adiposity and diabetes using 3T3-L1 models. Ad vector-mediated RNAi for PPARgamma should also be useful to clarify the biological role of the PPARgamma pathway in various tissues in addition to adipose and for therapeutic application to a variety of diseases, including adiposity and diabetes.
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http://dx.doi.org/10.1016/j.gene.2005.01.005DOI Listing
March 2005

Nuclear receptors as targets for drug development: the role of nuclear receptors during neural stem cell proliferation and differentiation.

J Pharmacol Sci 2005 Feb 11;97(2):171-6. Epub 2005 Feb 11.

Department of Biopharmaceutics, Graduate School of Pharmaceutical Science, Osaka University, Japan.

The fate of stem cells, such as neural stem cells and hematopoietic stem cells, depends on strictly regulated signaling events including activation of nuclear receptors, resulting in subsequent gene induction. Recently, we demonstrated that PPARgamma, a ligand-activated nuclear receptor, plays an important role in regulating the proliferation and differentiation of murine neural stem cell (NSC). NSC prepared from heterozygous PPARgamma-deficient mouse exhibited a slower growth rate compared with that of wild-type mouse, which was also demonstrated in PPARgamma-knockdown NSC that was generated by the lentiviral-vector-mediated RNA interference approach. These studies have important implications for understanding central nervous system functions and developing a therapy for neurodegenerative disorders. In this review, recent findings on stem cell biology, especially focusing on nuclear receptors in NSCs, including our current study, will be discussed.
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http://dx.doi.org/10.1254/jphs.fmj04008x3DOI Listing
February 2005

A single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin 12 induces remarkable anti-tumor and anti-metastatic activity in mice with Meth-A fibrosarcoma.

Biochem Biophys Res Commun 2005 Mar;328(4):1043-50

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

Cytokine-encoding viral vectors are considered to be promising in cancer gene immunotherapy. Interleukin 12 (IL-12) has been used widely for anti-tumor treatment, but the administration route and tumor characteristics strongly influence therapeutic efficiency. Meth-A fibrosarcoma has been demonstrated to be insensitive to IL-12 treatment via systemic administration. In the present study, we developed an IL-12-encoding fiber-mutant adenoviral vector (AdRGD-IL-12) that showed enhanced gene transfection efficiency in Meth-A tumor cells, and the production of IL-12 p70 in the culture supernatant from transfected cells was confirmed by ELISA. In therapeutic experiments, a single low-dose (2 x 10(7) plaque-forming units) intratumoral injection of AdRGD-IL-12 elicited pronounced anti-tumor activity and notably prolonged the survival of Meth-A fibrosarcoma-bearing mice. Immunohistochemical staining revealed that the IL-12 vector induced the accumulation of T cells in tumor tissue. Furthermore, intratumoral administration of the vector induced an anti-metastasis effect as well as long-term specific immunity against syngeneic tumor challenge.
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http://dx.doi.org/10.1016/j.bbrc.2005.01.061DOI Listing
March 2005

Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells.

Bioorg Med Chem Lett 2005 Feb;15(3):621-4

Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan.

The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (alphavbeta3 and alphavbeta5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-(beta)Ala)(2)Lys-PEG-(beta)Ala-Cys-NH(2)[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.
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http://dx.doi.org/10.1016/j.bmcl.2004.11.055DOI Listing
February 2005