Biomedical subjects

Mitsuru Hashida

Publications and source records attributed to Mitsuru Hashida.

At least 37 records · Page 2Linked to original sources

PubMed2006

Evaluation of proinflammatory cytokine production induced by linear and branched polyethylenimine/plasmid DNA complexes in mice.

The purpose of this study was to evaluate the cytokine response induced by linear and branched polyethylenimine (PEI)/plasmid DNA (pDNA) complex (polyplex) in relation to the ratio of PEI nitrogen and DNA phosphate (N/P ratio) of the polyplex, dose of pDNA, and structure and molecular weight of PEI, which are important for transfection efficacy of PEI polyplex. As a control, a N-[1-(2, 3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride/cholesterol liposome/pDNA complex (lipoplex) was selected for its high transfection efficacy in vivo. The concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha were much lower after the administration of polyplex than lipoplex irrespective of the N/P ratio, dose of pDNA, or structure and molecular weight of PEI, although these factors affected the transfection efficacy in vivo. We demonstrated that the amount of activated nuclear factor-kappaB, which contributes substantially to the production of cytokines, was comparable with the control (no treatment) level, and significantly less than that obtained with lipoplex. Although the production of proinflammatory cytokines (TNF-alpha, interferon-gamma, and interleukin-12) was reduced on the administration of the linear PEI polyplex, serum alanine aminotransferase levels were significantly enhanced by pDNA in a dose-dependent manner, suggesting that such hepatic damage is not induced by proinflammatory cytokines.

Alanine Transaminase↗

PubMed2006

Novel PEG-matrix metalloproteinase-2 cleavable peptide-lipid containing galactosylated liposomes for hepatocellular carcinoma-selective targeting.

In order to obtain an HCC-selective drug delivery system, a novel functional lipid, which is cleaved by the protease activity of matrix metalloproteinase-2 (MMP-2), was developed. The amino group of dioleoylphosphatidylethanolamine (DOPE) was conjugated with PEGylated MMP-2 substrate peptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln), and MMP-2-cleavable PEG-Peptide-DOPE (PEG-PD) was synthesized. When PEG-PD was incorporated in galactosylated liposomes (Gal-PEG-PD-liposomes), we expected that Gal-PEG-PD-liposomes would not be taken up by normal hepatocytes due to the steric hindrance effect, but would be activated around HCC cells by secreted MMPs. In the pretreatment by hMMP2 (1, 5, and 10mug/ml), an hMMP2 concentration-dependent higher uptake of Gal-PEG-PD-liposomes was observed in HepG2 cells, suggesting PEG-PD cleavage. In the presence of an excess of galactose, the uptake of Gal-PEG-PD-liposomes with hMMP2 was significantly inhibited, suggesting asialoglycoprotein receptor-mediated uptake of Gal-PEG-PD-liposomes following the PEG-PD cleavage. Pretreatment of Gal-PEG-PD-liposomes with the conditioned medium of B16BL6, which contained secreted MMPs, enhanced the binding to HepG2 cells, as in the case of hMMP-2 treatment. Moreover, the cytotoxicity of N(4)-octadecyl-1-beta-d-arabinofuranosylcytosine (NOAC) incorporated Gal-PEG-PD-liposomes was enhanced by hMMPs (5mug/ml) and its cytotoxicity was significantly reduced by the presence of an excess of galactose in HepG2 cells. In conclusion, Gal-PEG-PD-liposomes were successfully developed for novel HCC-selective targeting.

Antineoplastic Agents↗

PubMed2006

Improved anti-oxidant activity of superoxide dismutase by direct chemical modification.

Chemically modified derivatives of superoxide dismutase (SOD), i.e., cationized (Cat-SOD) and mannosylated SOD (Man-SOD), were designed to improve an ability of SOD to suppress reactive oxygen species (ROS)-mediated injury in the alveolar epithelium. To evaluate their effectiveness, an in vitro model of paraquat poisoning was developed with primary cultured rabbit alveolar type II cells. Despite a 5.6-fold higher cellular association than native SOD, Man-SOD did not protect cell injury due to paraquat following evaluation by MTT assay. In contrast, Cat-SOD exhibited a 140-fold higher cellular association than native SOD and greatly suppressed paraquat-induced cell injury, as well as lipid peroxidation. Incubation with 300 U/ml Cat-SOD for 2 h increased intracellular SOD activity 5.3-fold. The increase in intracellular SOD activity was significantly inhibited in the presence of cytochalasin B, an endocytosis inhibitor. Internalization of Cat-SOD was also confirmed by confocal laser scanning fluorescein microscopy. In addition, the protective effect of Cat-SOD against paraquat-induced cell injury was completely abolished by the presence of cytochalasin B. In conclusion, this study demonstrated that cationization of SOD greatly enhances its intracellular delivery and, as a consequence, produces a significant protective effect against ROS-mediated injury of the alveolar epithelium.

Animals↗

PubMed2006

Use of lipoplex-induced nuclear factor-kappaB activation to enhance transgene expression by lipoplex in mouse lung.

BACKGROUND: Although lipofection-induced TNF-alpha can activate nuclear factor kappaB (NF-kappaB), which, in turn, increases the transgene expression from plasmid DNA in which any NF-kappaB responsive element is incorporated, no attempts have been made to use such biological responses as NF-kappaB activation against a vector to enhance vector-mediated gene transfer. METHODS: A lipoplex composed of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium and cholesterol liposome and plasmid DNA encoding firefly luciferase under the control of the cytomegalovirus immediate early promoter (pCMV-Luc) was intravenously injected into mice. Luciferase activity as well as NF-kappaB activation in the lung were evaluated. Then, a novel plasmid DNA, pCMV-kappaB-Luc, was constructed by inserting 5 repeats of NF-kappaB-binding sequences into the pCMV-Luc. RESULTS: NF-kappaB in the lung was activated by injection of the lipoplex and its nuclear localization was observed. An injection of lipopolysaccharide 30 min prior to the lipofection further activated NF-kappaB. At the same time, the treatment significantly increased the transgene expression by lipoplex, suggesting a positive correlation between expression and NF-kappaB activity. Based on these findings, we tried to enhance the lipoplex-based transgene expression by using NF-kappaB activation. The lipoplex consisting of pCMV-kappaB-Luc showed a 4.7-fold increase in transgene expression in the lung compared with that with pCMV-Luc. CONCLUSIONS: We demonstrated that NF-kappaB activation by lipoplex can be used to enhance lipoplex-mediated transgene expression by inserting NF-kappaB-binding sequences into plasmid DNA. These findings offer a novel method for designing a vector for gene transfer in conjunction with biological responses to it.

Animals↗

PubMed2006

Enhanced DNA vaccine potency by mannosylated lipoplex after intraperitoneal administration.

BACKGROUND: Here we describe a novel DNA vaccine formulation that can enhance cytotoxic T lymphocyte (CTL) activity through efficient gene delivery to dendritic cells (DCs) by mannose receptor-mediated endocytosis. METHODS: Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. Mannosylated cationic liposomes (Man-liposomes) were prepared using cholesten-5-yloxy-N-{4-[(1-imino-2-D-thiomannosylethyl)amino]butyl}formamide (Man-C4-Chol) with cationic lipid. The potency of the mannosylated liposome/pCMV-OVA complex (Man-lipoplex) was evaluated by measuring OVA mRNA in CD11c+ cells, CTL activity, and the OVA-specific anti-tumor effect after in vivo administration. RESULTS: An in vitro study using DC2.4 cells demonstrated that Man-liposomes could transfect pCMV-OVA more efficiently than cationic liposomes via mannose receptor-mediated endocytosis. In vivo studies revealed that the Man-lipoplex exhibited higher OVA mRNA expression in CD11c+ cells in the spleen and peritoneal cavity and provided a stronger OVA-specific CTL response than intraperitoneal (i.p.) administration of the conventional lipoplex and intramuscular (i.m.) administration of naked pCMV-OVA, the standard protocol for DNA vaccination. Pre-immunization with the Man-lipoplex provided much better OVA-specific anti-tumor effect than naked pCMV-OVA via the i.m. route. CONCLUSIONS: These results suggested that in vivo active targeting of DNA vaccine to DCs with Man-lipoplex might prove useful for the rational design of DNA vaccine.

Animals↗

PubMed2006

Quantitative structure/activity relationship modelling of pharmacokinetic properties using genetic algorithm-combined partial least squares method.

Quantitative structure/activity relationship (QSAR) approaches have widely been applied to gain deeper understandings of the relationships between ADME parameters and molecular structure and properties. QSAR models for predicting ADME properties are required to cover structurally diverse compounds. In the present investigation, we describe application of genetic algorithm-combined partial least squares (GA-PLS) method to QSAR modelling of various ADME properties. By selecting an appropriate set of molecular descriptors automatically by the use of genetic algorithm, many ADME properties could be well-explained by simple molecular descriptors derived from 2-dimensional chemical structure.

Algorithms↗

PubMed2006

Basic fibroblast growth factor-binding peptide as a novel targeting ligand of drug carrier to tumor cells.

Drug systems targeting tumor cells using basic fibroblast growth factor (bFGF) have been widely reported. In this study, the peptide KRTGQYKLC (bFGFp), containing cysteine at the carboxyl termination of the bFGF-derived peptide, was applied as a novel ligand targeting tumor cells. bFGFp was conjugated with bovine serum albumin (BSA) and liposomes. The peptide was shown to inhibit the binding of bFGF to FGF receptor-1 (FGFR1). Interestingly, the binding study using surface plasmon resonance (SPR) assay revealed that the bFGFp-BSA was not bound to FGFR1, but was selectively bound to bFGF. Furthermore, the SPR assay showed that bFGFp-BSA is capable of binding to FGFR1 following the pretreatment with bFGF. The confocal microscopy study indicated that the uptake of bFGFp-BSA by NIH3T3 cells, which highly express FGFRs, was significantly enhanced by pretreatment with bFGF. Then, PEGylated liposomes containing bFGFp (bFGFp-liposome) were prepared by conjugating maleimide-PEG-PE with bFGFp. Following the pretreatment of bFGF, the uptake of bFGFp-liposomes by NIH3T3 cells was significantly enhanced. These results suggest that bFGFp-BSA and bFGFp-liposomes are taken by NIH3T3 cells via binding with bFGF. In addition, both bFGFp-BSA and bFGFp-liposomes had no effect on the proliferation of NIH3T3 cells. This strategy can be used as a novel system for targeting tumors highly expressing FGFRs without a proliferation effect.

Animals↗

PubMed2006

Effect of the particle size of galactosylated lipoplex on hepatocyte-selective gene transfection after intraportal administration.

The purpose of this study was to examine the effect of the size of galactosylated cationic liposome (Gal-liposome)/plasmid DNA complex (Gal-lipoplex) on hepatocyte-selective gene transfection after intraportal administration. pCMV-Luc was selected as a model plasmid DNA. After intraportal administration of Gal-lipoplex to mice, the hepatic and intrahepatic gene expression was evaluated. To evaluate the effect of size, three different sizes of Gal-liposome were prepared. The mean particle sizes of Gal-lipoplex were about 141, 179, and 235 nm, respectively. The hepatic transfection efficacy was significantly enhanced by increasing the size of Gal-lipoplex. However, the gene expression in liver parenchymal cells (PC) of Gal-lipoplex of about 141 nm in size was significantly higher than that in liver non-parenchymal cells (NPC). In contrast, gene expression in PC of Gal-lipoplex of about 235 nm in size was significantly lower than that in NPC. These results highlight the importance of the Gal-lipoplex size for hepatocyte-selective gene transfer in vivo. The information in this study will be valuable for the future use, design, and development of Gal-lipoplex for in vivo applications.

Animals↗

PubMed2006

Role of tyrosine and tryptophan in chemically modified serum albumin on its tissue distribution.

To investigate the effect of functional groups in bovine serum albumin (BSA) on its tissue distribution characteristics, tyrosine (Tyr) or tryptophan (Trp) residues of BSA were chemically modified by tetranitromethane (TNM) and 2-hydroxy-5-nitrobenzyl bromide (HNB), respectively. BSA was successfully modified with each reagent depending on the amount of the reagent added to the reaction mixture, and TNM- and HNB-modified BSA derivatives with different degrees of modification were obtained. Circular dichroism measurements showed that slight secondary and large tertiary changes were detectable as the degree of modification increased. After intravenous injection into mice, all synthetic BSA derivatives were eliminated very slowly from the systemic circulation. However, (111)In-TNM(6.6)- and (111)In-HNB(2.0)-BSA, derivatives with a high degree of modification, showed a slightly faster disappearance from the systemic circulation and slightly higher accumulation in the liver than (111)In-unmodified BSA. Pharmacokinetic analyses also demonstrated that the modification of Tyr or Trp residues on BSA had only marginal effects on tissue distribution. These results indicate that the Tyr and Trp residues have little effect on the tissue distribution characteristics of serum albumins, and that the specific modification of these residues may be a promising approach to designing sustained drug delivery systems using serum albumins.

2-Hydroxy-5-nitrobenzyl Bromide↗

PubMed2006

Intracellular trafficking is the important process that determines the optimal charge ratio on transfection by galactosylated lipoplex in HEPG2 cells.

The purpose of the present study was to gain insight into the major factors affecting transfection efficiency with galactosylated lipoplex in HepG2 cells. In this study, lipoplex and galactosylated lipoplex were examined at different charge ratios (- : +): 1.0 : 1.2, 1.0 : 2.3, 1.0 : 3.1, 1.0 : 4.7, and 1.0 : 7.0. The particle size and zeta potential of the both lipoplexes was dependent on the charge ratio. Cellular uptake was evaluated by using [(32)P]-labeled pCMV-Luc and this showed that the cellular uptake of galactosylated lipoplex was significantly higher than that of lipoplex at a charge ratio ranging from 1.0 : 2.3 to 1.0 : 7.0. As the charge ratio increased in both lipoplexes, the apparent cellular uptake increased. Transfection activity by galactosylated lipoplex was significantly higher than that by lipoplex except at a charge ratio of 1.0 : 7.0. The optimal charge ratio for transfection efficacy was 1.0 : 2.3 and transfection was reduced at higher charge ratios. Both lipoplexes exhibited no significant cytotoxicity at any charge ratio. In conclusion, it is suggested that intracellular trafficking, rather than the degree of uptake and cytotoxicity, is the important process that determines the optimal charge ratio of galactosylated lipoplex in HepG2 cells.

Biological Transport↗

PubMed2006

Influence of cholesterol composition on the association of serum mannan-binding proteins with mannosylated liposomes.

In our previous studies, serum mannan-binding protein (MBP) accelerated the uptake by cultured macrophages. The present study was initiated to investigate the kinetics of molecular interaction between mannosylated liposomes and MBP in more details and the effects of lipid composition on the interaction. The analysis was carried out by surface plasmon spectroscopy (SPR) methods, using rabbit serum MBP isolated by affinity chromatography. In SPR studies, neither conventional liposomes nor galactosylated liposomes indicated any interaction, but each mannosylated liposomes had a high response signal corresponding to molecular interaction with immobilized MBP. Association of mannosylated liposomes to serum MBP was not dependent on the lipid composition, suggesting a diffusion-controlled association. Dissociation of the mannosylated liposomes from serum MBP was extremely slow. DSPC/Chol/Man-C4-Chol (90 : 5 : 5, molar ratio) exhibited a slower dissociation rate than DSPC/Chol/Man-C4-Chol (60 : 35 : 5). Clustering of mannose residues on liposomal surfaces might be important in determining the binding affinity of mannosylated liposomes with MBP.

Animals↗

PubMed2006

Inhibition of tumour metastasis by targeted delivery of antioxidant enzymes.

Metastasis is one of the most harmful aspects of malignant neoplasm. Interaction of tumour cells with normal cells such as tissue macrophages may generate reactive oxygen species, which would affect various aspects of tumour metastasis. Reactive oxygen species cause damage to both tumour and normal cells and some of them, especially hydrogen peroxide, can also act as intracellular second messengers at sublethal concentrations to increase the transcription of various genes, which can then accelerate the proliferation of tumour cells in metastatic colonies. Therefore, eliminating hydrogen peroxide is one approach to inhibiting tumour metastasis. In this article, the roles of reactive oxygen species in tumour metastasis are reviewed, and the strategies to inhibit tumour metastasis by the targeted delivery of catalase, an enzyme that detoxifies hydrogen peroxide, are discussed.

Animals↗

PubMed2006

[Pharmaceutical device of IFN].

Recently, new types of interferon (IFN) agents, such as pegylated IFN (PEG-IFN) and consensus IFN, have been developed for strong therapeutic effect and used in clinical stage. PEG-IFN, which is the conjugation of IFN and polyethylene glycol, was designed to long circulate in blood and decrease antibody production. Consensus IFN was designed to combine amino acid frequently appeared in IFN-alpha subtypes. Although they improved IFN therapy, it is also important to pay attention to side effect. Characteristics of new type IFN agents were summarized focused on their pharmaceutical designs.

Amino Acid Sequence↗

PubMed2005

Induction of apoptosis in A549 human lung cancer cells by all-trans retinoic acid incorporated in DOTAP/cholesterol liposomes.

All-trans retinoic acid (ATRA) has been shown to exert anti-cancer activities in a number of types of cancer cells. However, it has been reported that many NSCLC exhibited resistance to ATRA treatment. In the present study, we hypothesized that intracellular delivery of ATRA would overcome the ATRA resistance in A549 cells. Here, we investigated the induction of apoptosis by ATRA incorporated in cationic liposomes composed of DOTAP/cholesterol in A549 human lung cancer cells, which are insensitive (resistant) to the growth inhibitory effects of ATRA. The zeta potentials of DOTAP/cholesterol liposomes and DSPC/cholesterol liposomes were about +50 and -3 mV. In A549 cells, [(3)H]ATRA incorporated in DOTAP liposomes showed increased cellular association compared with [(3)H]ATRA or [(3)H]ATRA incorporated in DSPC/cholesterol liposomes. ATRA incorporated in DOTAP/cholesterol liposomes showed much higher cytotoxic effects and apoptosis-inducing activity compared with ATRA or ATRA incorporated in DSPC/cholesterol liposomes. The enhanced expression of TIG3 mRNA tumor suppressor gene by ATRA incorporation into DOTAP/cholesterol liposomes might partly explain the mechanism of enhanced cytotoxicity and/or apoptosis. These observations provide valuable information to help in the design of differentiation therapy by ATRA in non-small cell lung carcinoma.

Apoptosis↗

PubMed2005

Liver targeting of catalase by cationization for prevention of acute liver failure in mice.

To achieve hepatic delivery of CAT for the prevention of CCl4-induced acute liver failure in mice, two types of cationized CAT derivatives, HMD- and ED-conjugated CAT, were developed. Slight structural changes occurred during cationization and the number of increased free amino groups was 3.1 in HMD-CAT and 13.6 in ED-CAT. 111In-cationized CAT derivatives showed an increased binding to HepG2 cells, and were rapidly taken up by the liver. H2O2-induced cytotoxicity in HepG2 cells was significantly prevented by preincubation of the cells with cationized CAT derivatives. A bolus intravenous injection of the cationized CAT derivatives reduced the hepatotoxicity induced by CCl4 in mice. The ED-CAT, which showed more rapid and greater binding to the liver than the HMD-CAT, exhibited more beneficial effects as far as all the parameters examined (serum GOT, GPT, LDH and hepatic GSH) were concerned, suggesting that a high degree of cationization is effective in delivering CAT to the liver to prevent CCl4-induced hepatotoxicity. These results suggest that cationized CAT derivatives are effective in preventing acute liver failure, and ED-based cationization is a suitable method for developing liver-targetable cationized CAT derivatives, because it provides CAT with a high degree of cationization and a high remaining enzymatic activity.

Animals↗

PubMed2005

Inhibition of metastatic tumor growth by targeted delivery of antioxidant enzymes.

To develop effective anti-metastatic therapy, targeted or sustained delivery of catalase was examined in mice. We found that mouse lung with metastatic colonies of adenocarcinoma colon26 cells exhibited reduced catalase activity. The interaction of the tumor cells with macrophages or hepatocytes generated detectable amounts of ROS, and increased the activity of matrix metalloproteinases. Hepatocyte-targeted delivery of catalase was successfully achieved by galactosylation, which was highly effective in inhibiting the hepatic metastasis of colon26 cells. PEGylation, which increased the retention of catalase in the circulation, effectively inhibited the pulmonary metastasis of the cells. To examine which processes in tumor metastasis are inhibited by catalase derivatives, the tissue distribution and proliferation of tumor cells in mice was quantitatively analyzed using firefly luciferase-expressing tumor cells. An injection of PEG-catalase just before the inoculation of melanoma B16-BL6/Luc cells significantly reduced the number of the tumor cells in the lung at 24 h. Daily dosing of PEG-catalase greatly inhibited the proliferation of the tumor cells, and increased the survival rate of the tumor-bearing mice. These results indicate that targeted or sustained delivery of catalase to sites where tumor cells metastasize is a promising approach for inhibiting metastatic tumor growth.

Animals↗

PubMed2005

Intracellular distribution of NFkappaB decoy and its inhibitory effect on TNFalpha production by LPS stimulated RAW 264.7 cells.

Nuclear factor kappa B (NFkappaB) is a transcriptional factor for the expression of many cytokines that are involved in the pathogenesis of inflammatory diseases. Unstimulated NFkappaB sequestered in the cytoplasm bound to inhibitory proteins is called IkappaBs. Many activators of NFkappaB cause degradation of IkappaB proteins and free NFkappaB can enter the nucleus and induce gene expression. In this study, we analyzed the relationship between the intracellular distribution and pharmacological effect of NFkappaB decoy in RAW 264.7 cells. Most of the fluorescent labeled NFkappaB decoy was observed in the cytoplasm both with or without cationic transfection without LPS stimulation. Furthermore, under LPS stimulation, most of NFkappaB decoy was also observed in the cytoplasm. However, NFkappaB decoy effectively inhibited the production of TNFalpha in RAW 264.7 cells. The inhibitory effect of TNFalpha production by NFkappaB decoy transfected by cationic liposomes was much stronger than that by naked NFkappaB decoy, because the amount of cellular association of NFkappaB transfected by cationic liposome decoy was 7 times higher than that of naked NFkappaB decoy. This information is of great value for the design of NFkappaB decoy carrier systems.

Animals↗

PubMed2005

The role of dioleoylphosphatidylethanolamine (DOPE) in targeted gene delivery with mannosylated cationic liposomes via intravenous route.

We have previously reported that mannosylated cationic liposome consisting with the mannosylated cationic cholesterol derivative Man-C4-Chol (Man) and dioleoylphosphatidylethanolamine (DOPE) (Man/DOPE) could deliver DNA to the liver by intravenous administration via mannose receptor-mediated endocytosis, however, rapid degradation in lysosomes might be a rate-limiting step in its gene transfection. In this study, we tried to evaluate the role of DOPE in in vivo gene transfer by comparing its transfection efficacy with mannosylated liposomes composed of Man and dioleoylphosphatidylcholine (DOPC) (Man/DOPC). In vitro studies showed that the cellular association of both liposome/pCMV-Luc complexes was almost the same, although Man/DOPE complex showed about 10-fold higher transfection activity than Man/DOPC complex. After intraportal administration into mice, Man/DOPE complex showed higher gene expression than Man/DOPC complex, suggesting that DOPE improves intracellular trafficking in target cells under in vivo conditions. An intravenous administration study demonstrated that Man/DOPE complex was accumulated in the liver more efficiently and achieved a higher gene expression in the liver than Man/DOPC complex. Thus, we conclude that the property of DOPE in mannosylated liposomes contributes to the efficient gene expression in the target site through enhanced distribution to the target site and intracellular sorting in the target cells under in vivo conditions.

Animals↗