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Biomedical subjects

H Harashima

Publications and source records attributed to H Harashima.

At least 19 recordsLinked to original sources
PubMed2006

Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid.

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

Animals↗
PubMed2004

Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy.

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

Animals↗
PubMed2004

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation.

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

3T3 Cells↗
PubMed2003

Two cases with transient lipoprotein lipase (LPL) activity impairment: evidence for the possible involvement of an LPL inhibitor.

UNLABELLED: Two independent severe hypertriglyceridemic infants with transiently impaired lipoprotein lipase (LPL) activity were observed and the causes were explored. Both infants were female, born prematurely with low birth weight and developed hypertriglyceridemia (Fredrickson type V hyperlipidemia: high VLDL and low LDL/HDL) a few months after birth. While mass levels of their post-heparin plasma LPL and apoprotein C-II (apo C-II), a physiological activator of LPL, were normal, their post-heparin plasma LPL activities were remarkably impaired. Both of their mothers' post-heparin plasma LPL activities were slightly or moderately impaired as well, without a decrease in the LPL mass level. No mutations in the genes for LPL and apo C-II were detected in either patient. In an in vitro study with their serum at onset, we could not detect any distinct circulating inhibitors for LPL. There was no data supporting infection or autoimmune diseases, which might have an impact on LPL activity, during the follow-up period. Levels of their plasma triglyceride (TG) and total cholesterol (TC) were decreased quickly by a dietary intervention with medium-chain triglyceride (MCT) milk and kept normal even after stopping the intervention at around age 1 year. However, their low post-heparin LPL activity persisted and returned to normal at around age 2 years. Their low HDL cholesterol levels persisted even after recovery of the TG and TC levels, although lecithin:cholesterol acyltransferase (LCAT) and cholesterol-ester-transfer protein (CETP), two key enzymes of HDL metabolism, were normal throughout the course. The exact reasons why their post-heparin LPL activities were impaired for a certain period and why their HDL cholesterol levels have remained low are still unclear. CONCLUSION: Transiently impaired LPL activity with no defect in LPL enzyme induced severe hypertriglyceridemia in infants. The transient occurrence of inhibitor(s) for LPL was proposed.

Child↗
PubMed2002

Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells.

We prepared a series of linearized DNA duplexes of various lengths in order to examine the effects of topology and the size of exogenous, plasmid-derived DNAs on transgene expression. These linearized DNA duplexes were capped at each end with a highly stable loop (5'-GCGAAGC-3') to produce a dumbbell-shaped construct that is refractory to exonuclease digestion in comparison to the analogous uncapped DNA duplexes. Intranuclear microinjection of the DNA dumbbells into simian COS-7 cells allowed the expression of the green fluorescent protein (GFP) gene on the linearized molecules, which was expressed five- to 10-fold more than that on the circular DNA of the same size. In addition, the expression by the dumbbell DNA was higher than that by the circular plasmid for at least 14 days. Interestingly, the size of the dumbbell DNA affected the transgene expression upon their microinjection into cell nuclei. The GFP expression efficiency increased with decreasing DNA size below a DNA size of 5.7 kb. The effects of topology and size on the expression of DNAs transfected with cationic lipids are similar to those of DNAs microinjected into cell nuclei. In contrast, microinjection into the cytosol showed the inverse size dependency over a range of 2.3 to 9.4 kb. Thus, transcription of a transgene in the nucleus, but not endocytosis or nuclear entry, was influenced by the exogenous DNA structure, and this was the primary determinant of transgene expression upon transfection under our conditions. These results indicate that small, linearized DNA duplexes that are end-capped with a highly stable loop (dumbbell-shaped DNA) would be very useful for nonviral gene therapy.

3T3 Cells↗
PubMed2001

Quantitative studies on the nuclear transport of plasmid DNA and gene expression employing nonviral vectors.

The nuclear membrane is the main barrier to nonviral gene delivery. Thus, in the case of nondividing cells, a device for nuclear delivery of exogenous DNA is necessary. In addition, to precisely evaluate the efficacy of various plasmid modifications and/or nonviral vectors, it is necessary to measure, not only gene expression but also the amount of delivered plasmid DNA into the subcellular compartment, particularly the nucleus. Moreover, it is also necessary to examine effects of the state of the plasmid DNA in the nucleus or various modifications of the plasmid DNA on the process after nuclear transport, i.e., transcription. Here, we address the issues of (1) the efficient delivery of genetic materials using a nuclear localization signal (NLS), (2) the quantitative evaluation of plasmid DNA delivered to the nucleus and the relationship between the amount of plasmid DNA delivered into the nucleus and gene expression, and (3) methods for evaluating of the effect of the state of plasmid DNA on transcription in vitro.

Active Transport, Cell Nucleus↗
PubMed2001

Intracellular trafficking and transgene expression of viral and non-viral gene vectors.

The objective of this review is to summarize the critical steps in intracellular trafficking and the principal factors involved in transgene expression by a comparison of viral and non-viral gene vectors. Intracellular trafficking of viral and non-viral gene vectors are reviewed kinetically as well as from the mechanistic point of view. The peptide-dependent specific mechanism of endosomal escape by viral vectors is compared with the non-specific mechanism of non-viral vectors. Regarding the nuclear transport of DNA, a number of recently developed strategies in non-viral vectors such as the application of nuclear localization signals or cell specific transcription factors are summarized in comparison with viral nuclear gene delivery. The molecular mechanisms of transcription and the translation of delivered genes to nucleus are also summarized in view of drug delivery systems. This information is intended to serve as a basis for developing a new gene delivery system for both viral and non-viral gene vectors. Optimizing the gene delivery system by integrating this intracellular trafficking as well as transgene expression will be required in order to develop an efficient and an safe gene delivery system.

Animals↗
PubMed2001

Hydrolysis of oxidized nucleotides by the Escherichia coli Orf135 protein.

To examine the possibility that the Orf135 protein of Escherichia coli functions as a hydrolyzing enzyme for a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate), we purified the recombinant Orf135 protein and incubated it with oxidized deoxynucleotides. Of the nucleotides tested, 2-hydroxydeoxyadenosine 5'-triphosphate, and somewhat less efficiently, 8-hydroxydeoxyguanosine 5'-triphosphate, were hydrolyzed by this protein. These damaged deoxynucleotides elicit transversion mutations in E. coli (Inoue, M., Kamiya, H., Fujikawa, K., Ootsuyama, Y., Murata-Kamiya, N., Osaki, T., Yasumoto, K., Kasai, H. (1998) J. Biol. Chem. 273, 11069-11074). These results suggest that this protein may be involved in the prevention of mutations induced by these oxidized deoxynucleotides.

DNA Damage↗
PubMed2001

Effect of cholesterol content in activation of the classical versus the alternative pathway of rat complement system induced by hydrogenated egg phosphatidylcholine-based liposomes.

Liposomes composed of hydrogenated egg phosphatidylcholine (HEPC) and cholesterol (CHOL) were found to activate the rat complement (C) system in a CHOL content-dependent manner. Liposomes containing 22 or 33 mol% CHOL activated the C system in a Ca(2+)-dependent manner, suggesting that C activation occurred via the classical pathway. Liposomes containing 44 mol% CHOL activated the C system in a Ca(2+) independent manner, suggesting that C activation occurred via the alternative pathway. The CHOL content appeared to dictate the pathway by which the C system was activated. This C activation was inhibited by removal of serum component(s), which adsorb to the liposomes. Activation of the alternative pathway, induced by the liposomes, was reduced by the depletion of IgG and IgM, whereas the classical pathway activation was reduced by the depletion of IgG, but not IgM. In addition, the removal of adsorbed serum component(s) by treatment with 44 mol% CHOL-containing liposomes decreased serum IgG and IgM levels that adsorb to the same liposomes, whereas the removal of adsorbed serum component(s) by treatment with 22 mol% CHOL-containing liposomes only slightly decreased serum IgG levels, which adsorbs to the same liposomes. Collectively, both IgG and IgM, which are specifically adsorbed to the liposomes in a CHOL-content dependent manner, were responsible for C activation via the alternative pathway induced by the 44 mol% CHOL containing liposomes. IgG alone would be partially responsible for C activation via the classical pathway induced by 22 or 33 mol% CHOL-containing liposomes. The discovery of this unique C-activating property of liposomes will be of value in attempts to decipher the underlying mechanism of C activation by providing a useful model membrane system.

Animals↗
PubMed2001

The complement system enhances the clearance of phosphatidylserine (PS)-liposomes in rat and guinea pig.

In this study, we investigated the contribution of the complement system to the biodistribution of phosphatidylserine (PS)-containing liposomes in rat and guinea pig. It appeared that the inclusion of PS in the liposome formulation accelerates the rate of liposome uptake by liver, resulting in rapid elimination of the liposomes from blood circulation. Pretreatment with K76COOH (K76), an anti-complement agent, decreased the rapid uptake of PS-containing liposomes by guinea pig liver, resulting in increasing blood concentration of the liposomes. Significant complement-dependent liposome destabilization was observed in vitro in both animals, whereas the complement-dependent destabilization in vivo was likely only a part of the process of the clearance of the PS-containing liposomes. This discrepancy suggests that the rate of complement-dependent liposome uptake by liver is much faster than the rate of complement-dependent liposome destabilization in vivo. Pretreatment of K76 dramatically inhibited the binding of C3 fragments, one of dominant opsonins, to PS-containing liposomes in guinea pig under both in vivo and in vitro conditions. This finding suggests that the C3 fragments in the system are responsible for the clearance of the PS-containing liposomes in guinea pig. In rat, in contrast to guinea pig, in vivo binding of C3 fragments was not inhibited by K76-pretreatment, while in vitro binding was inhibited. This discrepancy may be due to different experimental conditions between in vitro and in vivo assay. Nevertheless, based on the observations in this study, the complement components are most likely involved in the clearance of the PS-containing liposomes in rat. Taken together, the activity of PS in enhancing the liposome clearance appears to be mediated by the complement components, presumably C3 fragments, in both guinea pig and rat. This is a first report showing the mechanism on the hepatic uptake of the PS-containing liposomes in guinea pig.

Animals↗
PubMed2001

Intracellular control of gene trafficking using liposomes as drug carriers.

The objective of this review is to summarize some of the critical barriers in gene delivery and recent progress in overcoming such barriers using non-viral carrier systems. Receptor-mediated endocytosis is generally considered to be a principal entering pathway. Therefore, endosomal escape is an essential step for achieving efficient transfection. The nuclear membrane is also a critical barrier in gene delivery and the application of the nuclear localization signal is discussed, based on recent strategies. It is essential to optimize the carrier system, in order to enhance the transfection ability equivalent to a viral system. The importance of developing an intracellular pharmacokinetic model of genes is emphasized in the optimization of non-viral carrier systems.

Animals↗
PubMed2001

Species difference in correlation between in vivo/in vitro liposome-complement interactions.

The objective of this study was to investigate the correlation between in vitro and in vivo liposome-complement interactions. Third component of the complement (C3) fragments associated with hydrogenated egg phosphatidylcholine (HEPC)-based liposomes in vivo and complement-dependent destabilization in vitro were determined as an indication of liposome-complement interaction in vivo and in vitro, respectively. C3 fragments on the liposomes were detected in both rats and guinea pigs. Pretreatment with K76COOH (K76), a complement inactivating agent, reduced the binding of C3 fragments. These findings indicated that the liposomes remarkably activated the complement system in both animals in vivo. Interestingly, significant complement-dependent liposome destabilization was observed in rat serum, but not in guinea pig serum, indicating that the liposomes activated the complement system in rats, but not in guinea pigs in vitro. Taken together, it is apparent that in vitro complement activation by the liposomes is not in agreement with in vivo complement activation in ginea pigs. This discrepancy in the liposome-complement interaction would suggest the need for further investigation to utilize the information obtained from the liposome-complement interaction to predict in vivo behavior of the liposomes.

Animals↗
PubMed2001

Interactions of liposomes with cells in vitro and in vivo: opsonins and receptors.

A number of studies have appeared recently on the underlying mechanisms of liposome-cell interactions under in vitro conditions, in which isolated cell populations or cell lines were used. However, our knowledge of how liposomes interact with cells and the parameters that influence this in vivo is limited. We will summarize and discuss the relevant studies on this matter in this article. In addition, researchers in this field have long been aware of the interaction of liposomes with blood (or serum/plasma) proteins in vivo and their potential role in the process of the clearance of liposomes from the circulation. Some of the 'opsonizing' proteins, such as complement components, immunoglobulins, which enhance the interactions of liposomes with 'phagocytic cells' have been identified. However, the issue of which types of opsonins determine the fate of liposomes in vivo and how liposomal physicochemical properties such as size, charge and fluidity play an important role in the process of liposome clearance is not clear. Our own observations of one of opsonins, complement component are reviewed herein. As opposed to the fate of conventional liposomes, we briefly touch on the interaction of surface-modified liposomes, which are designed to avoid interactions with blood proteins and/or cells (sterically stabilized liposomes, long-circulating liposomes) and to actively target specific cells or tissues (targeted liposomes: immunoliposomes). Blood proteins such as opsonins are not usually thought to play an important role in the clearance of such liposomes.

Antineoplastic Agents↗
PubMed2000

Biodistribution of liposomes and C3 fragments associated with liposomes: evaluation of their relationship.

The biodistribution of liposomes with two different kind phospholipids (hydrogenated egg phosphatidylcholine and egg phosphatidylcholine) plus cholesterol (CHOL) were investigated after intravenous administration to rats. Elimination of liposomes from blood circulation was affected by the lipid composition. It appeared that the inclusion of CHOL in liposomes accelerates the rate of liposome uptake by liver, resulting in rapid elimination of liposomes. The amount of C3 fragments bound to liposomes was quantitatively determined to assess the contribution of the complement system to liposome accumulation into organs and liposome destabilization in vivo and in vitro. The amount of bound C3 fragments was directly proportional to CHOL content, and the amount was also proportional to the CLh, CLs as well as CLrel. This relationship suggests that the complement system is responsible for the elimination of liposomes from blood circulation, presumably as a consequence of opsonization by C3 fragments and assembly of membrane attack complex (MAC) onto liposomes. In addition, substitution of cholesteryl methyl ether into the liposome formulation for CHOL significantly diminished not only the binding of C3 fragments but also the CLh, CLs and CLrel, resulting in increased mean resident time (MRT) of the liposomes. This result suggests that the hydroxyl-group on CHOL is a binding site for C3 fragments on the liposomes and that CHOL in a liposome formulation promotes the accumulation of liposomes into the liver and spleen, probably due to their uptake by phagocytic cells, and impairs the stability of the liposomes in blood circulation, via a mechanism involving the complement system.

Animals↗
PubMed2000

[Media for 21st century--towards human communication media].

Today, with the approach of the 21st century, attention is focused on multi-media communications combining computer, visual and audio technologies. This article discusses the communication media target and the technological problems constituting the nucleus of multi-media. The communication media is becoming an environment from which no one can escape. Since the media has such a great power, what is needed now is not to predict the future technologies, but to estimate the future world and take to responsibility for future environments.

Communications Media↗
PubMed1999

Pharmacokinetic/pharmacodynamic modeling of antitumor agents encapsulated into liposomes.

Pharmacokinetic/pharmacodynamic (PK/PD) modeling of antitumor agents has been developed for doxorubicin (DOX) in order to predict the optimum conditions for a drug carrier to maximize the antitumor effect. A PK model was constructed for free and liposomal doxorubicin using a hybrid model wherein the disposition in the whole body is described by compartment models, which were linked to the tumor compartment via the blood flow rate. The PD model for doxorubicin was described by a cell-kill kinetic model, which represents the number of tumor cells quantitatively, as a function of the free concentration of doxorubicin in the tumor compartment. The influence of each parameter on the antitumor effects was examined by sensitivity analysis based on the PK/PD model, which clearly showed the importance of optimizing the release rate of DOX from liposomes. The validity of the model has been tested using animal experiments. Preliminary simulations were also performed for humans after scaling up the PK/PD model from rodents to humans. The optimum conditions in the rate of drug release from liposomes were different for rodents vis-a-vis humans, which indicates the limitations involved in extrapolating optimum conditions for experimental animals to those for humans.

Journal Article↗