Shigeru Kawakami

Mannose receptor-mediated gene transfer into macrophages using novel mannosylated cationic liposomes.

A novel mannosylated cholesterol derivative, cholesten-5-yloxy-N-(4-((1-imino-2-β-D-thiomannosyl-ethyl)amino)butyl) formamide (Man-C4-Chol), was synthesized in order to perform mannose receptor-mediated gene transfer with liposomes. Plasmid DNA encoding luciferase gene (pCMV-Luc) complexed with liposomes, consisting of a 6:4 mixture of Man-C4-Chol and dioleoylphosphatidylethanolamine (DOPE), showed higher transfection activity than that complexed with 3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol)/DOPE(6:4) and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/DOPE(1:1) liposomes in mouse peritoneal macrophages. The presence of 20 mM mannose significantly inhibited the transfection efficiency of pCMV-Luc complexed with Man-C4-Chol/DC- Chol/DOPE(3:3:4) and Man-C4-Chol/DOPE(6:4) liposomes. High gene expression of pCMV-Luc was observed in the liver after intravenously injecting mice with Man-C4-Chol/DOPE(6:4) liposomes, whereas DC-Chol/DOPE(6:4) liposomes only showed marked expression in the lung. The gene expression with Man-C4-Chol/DOPE(6:4) liposome/ DNA complexes in the liver was observed preferentially in the non-parenchymal cells and was significantly reduced by predosing with mannosylated bovine serum albumin. The gene expression in the liver was greater following intraportal injection. These results suggest that plasmid DNA complexed with mannosylated liposomes exhibits high transfection activity due to recognition by mannose receptors both in vitro and in vivo.

In vivo gene delivery to the liver using novel galactosylated cationic liposomes

AbstractPurpose. The purpose of this study is to elucidate the in vivo genetransfer for galactosylated liposomes containingcholesten-5-yloxy-N-(4-((1-imino-2-β-D-thiogalactosylethyl)amino)butyl)formamide(Gal-C4-Chol)in relation to lipid composition and charge ratio. Methods. Galactosylated cationic liposomes containingN-]1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride(DOTMA),Gal-C4-Chol and cholesterol(Chol), and similar liposomes were prepared.Plasmid DNA complexed with a galactosylated liposome preparationwas injected intraportally into mice. The mice were sacrificed after 6hours. The tissues were subjected to luciferase assay. Results. A markedly higher gene expression in the liver followinginjection of plasmid DNA that has been complexed withDOTMA/Chol/Gal-C4-Chol(1:0.5:0.5) and DOTMA/Gal-C4-Chol(1:1)liposomes was observed. The effect was one order of magnitude higherthan naked DNA and DOTMA/Chol(1:1) liposomes. Pre-exposing withgalactosylated bovine serum albumin significantly reduced the hepaticgene expression. By comparison, the gene expression for galactosylatedcationic liposomes containing3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol,Gal-C4-Chol and dioleoylphosphatidylethanolamine was 10 times lower.As far as the charge ratio of DOTMA/Chol/Gal-CA-Chol(1:0.5:0.5) liposomesto plasmid DNA(1.6-7.0) was concerned, complexes with charge ratiosof 2.3-3.1 produced maximal gene expression in the liver. Whereas,higher ratios resulted in enhanced expression in the lung. Conclusions. By optimizing lipid composition and charge ratio,galactosylated liposome/DNA complexes allow superior in vivo genetransfection in the liver via asialoglycoprotein receptor-mediatedendocytosis.

Molecular weight-dependent gene transfection activity of unmodified and galactosylated polyethyleneimine on hepatoma cells and mouse liver

To optimize a receptor-mediated and cell-selective gene transfer with polyethyleneimine (PEI)-based vector, we synthesized three galactosylated PEIs (Gal-PEI) with different molecular weights (PEI(1800), PEI(10,000), and PEI(70,000)) and investigated their potential as a targetable vector to asialoglycoprotein receptor-positive cells. All PEI derivatives formed complexes with plasmid DNA (pDNA), whereas the particle size of the complex became smaller on increasing the molecular weight of PEI. Transfection efficiency in HepG2 cells with PEI was highest with PEI(1800); efficiency was next highest with PEI(10,000), although the cellular association was similar. After galactosylation, Gal(19)-PEI(10,000)/pDNA and Gal(120)-PEI(70,000)/pDNA showed considerable agglutination with a galactose-recognizing lectin, but Gal(9)-PEI(1800) did not, suggesting that galactose units on the Gal(9)-PEI(1800)-pDNA complex are not sufficiently available for recognition. Gal(19)-PEI(10,000)-pDNA and Gal(120)-PEI(70,000)-pDNA complexes showed galactose-inhibitable transgene expression in HepG2 cells. Transfection efficiency was greatest with Gal(19)-PEI(10,000)/pDNA, a result that highlights the importance of obtaining a balance between the cytotoxicity and the transfection activity, both of which are found to be a function of the molecular weight of PEI. After intraportal injection, however, Gal(153)-PEI(70,000)/pDNA having a low N/P ratio was most effective, suggesting that additional variables, such as the size of the complex, are important for in vivo gene transfer to hepatocytes.

Strategies for In Vivo Delivery of siRNAs

RNA interference (RNAi) is a post-transcriptional gene-silencing mechanism that involves the degradation of messenger RNA in a highly sequence-specific manner. Double-stranded small interfering RNA (siRNA), consisting of 21–25 nucleotides, can induce RNAi and inhibit the expression of target proteins. Therefore, siRNA is considered a promising therapeutic for treatment of a variety of diseases, including genetic and viral diseases, and cancer. Clinical trials of siRNA are ongoing or have been planned, although some issues need to be addressed. For example, cellular uptake of naked siRNA is extremely low due to its polyanionic nature. Furthermore, siRNA is easily degraded by enzymes in blood, tissues, and cells. Several types of chemically modified siRNA have been produced and investigated to improve stability; these have involved modification of the siRNA backbone, the sugar moiety, and the nucleotide bases of antisense and/or sense strands. Because the accumulation at the target site after administration is extremely low, even if stability is improved, an effective delivery system is required to induce RNAi at the site of action. Delivery strategies can be categorized into physical methods, conjugation methods, and drug delivery system carrier-mediated methods. Physical techniques can enhance siRNA uptake at a specific tissue site using electroporation, pressure, mechanical massage, etc. Terminal modification of siRNAs can enhance their resistance to degradation by exonucleases in serum and tissue. Moreover, modification with a suitable ligand can achieve targeted delivery. Several types of carrier for drug delivery have been developed for siRNA in addition to traditional cationic liposome and cationic polymer systems. Ultrasound and microbubbles or liposomal bubbles have also been used in combination with a carrier for siRNA delivery. New materials with unique characteristics such as carbon nanotubes, gold nanoparticles, and gold nanorods have attracted attention as innovative carriers for siRNA.

Biodistribution characteristics of mannosylated, fucosylated, and galactosylated liposomes in mice

The in vivo disposition behavior and pharmacokinetic characteristics of galactosylated (Gal), mannosylated (Man) and fucosylated (Fuc) liposomes were compared in this study. For the preparation of the glycosylated liposomes, cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiogalactosyle thyl)amino)a lkyl)formamide (Gal-C4-Chol) (Kawakami et al., Biochem. Biophys. Res. Commun. 252 (1998) 78-83) and its mannosylated and fucosylated derivatives (Man-C4-Chol and Fuc-C4-Chol, respectively) were synthesized. The glycosylated liposomes are composed of distearoylphosphatidylcholine (DSPC), cholesterol (Chol), and Gal-C4-Chol (or Man-C4-Chol or Fuc-C4-Chol) with the molar ratio of 60:35:5. After intravenous injection in mice, these three types of [(3)H]cholesteryl hexadecyl ether-labeled glycosylated liposomes were rapidly eliminated from the circulating blood and preferentially recovered in the liver. In contrast, DSPC/Chol (60:40) liposomes without glycosylation were retained for a long time in the circulating blood. The uptake ratios by parenchymal cells (PC) and nonparenchymal cells (NPC) (PC/NPC ratios) for 0.5% Gal, Man and Fuc liposomes were found to be 15.1, 0.6 and 0.2, respectively. The effect of predosing glycosylated proteins and liposomes on the hepatic uptake of 0.5% (3)H-labeled Gal, Man, and Fuc liposomes was investigated and the results support the conclusion that Gal, Man, and Fuc liposomes are taken up by the liver via asialoglycoprotein receptors in PC, mannose receptors in NPC, and fucose receptors in NPC, respectively. Interestingly, Gal liposomes were taken up by NPC rather than by PC at a high dose (5%). Together with the finding that 5% Gal liposomes inhibit the hepatic uptake of (3)H-labeled Fuc liposomes, this suggests that Gal-liposomes administered at a high dose will also be taken up by fucose receptors in NPC, that are considered to act as galactose particle receptors.

Efficient Gene Transfection by Histidine-Modified Chitosan through Enhancement of Endosomal Escape

Chitosan has the potential to be a biocompatible gene carrier. However, the transfection efficiency of chitosan is low because of the slow endosomal escape rate. The buffering capacity of histidine in the endosomal pH range would help the escape of plasmid DNA (pDNA) from endosomes. In this study, histidine was introduced into chitosan to improve the transfection efficiency. Chitosan and histidine were linked by disulfide bonds provided by 2-iminothiolane and cysteine. The complexes were prepared by mixing chitosan or histidine-modified chitosan with plasmid DNA. A broader buffering range of histidine-modified chitosan was observed, and the cellular uptake of histidine-modified chitosan/pDNA complexes was higher than that of chitosan/pDNA complexes. Although chitosan/tetramethylrhodamine (TMR)-pDNA complexes were trapped in the vesicles in cytosol, TMR-pDNA carried by histidine-modified chitosan was more widely distributed in the cytosol. This result suggests that histidine can help pDNA escape from endosomes with the help of the high buffering capacity. The gene expression of histidine-modified chitosan/pDNA complexes was higher than that of chitosan/pDNA complexes. These results suggest that histidine modification improves the transfection efficiency of chitosan.

Designing Dendrimers for Drug Delivery and Imaging: Pharmacokinetic Considerations

ABSTRACTDendrimers have well-organized high branches with a layered architecture providing a series of versatile chemical modification for various purposes. Consequently, this dendrimer nanotechnology explores a new promising class of nanoscale carriers for therapeutic drugs and imaging reagents using passive and active targeting approaches. By controlling dendritic structures, the biological fate of dendrimer/dendrimer-based drugs can be significantly altered based on their intrinsic physicochemical properties, including the hydrophilicity of the unit molecules, particle size, surface charge, and modification. Accordingly, pharmacokinetic aspects play an important role in the design and development of dendrimer systems for successful in vivo application and clinical translation. This review focuses on the recent progress regarding dendritic architectures, structure-related toxicity, and critical factors affecting the pharmacokinetics and biodistribution of dendrimer/dendrimer-based drugs. A better understanding of the basic aspects of dendritic systems and their pharmacokinetics will help to develop a rationale for the design of dendrimers for the controlled delivery of drugs and imaging reagents for therapeutic or diagnostic purposes.

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)-α 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-κB, 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-α, interferon-γ, 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.

piggyBac Transposon-mediated Long-term Gene Expression in Mice

Transposons are promising systems for somatic gene integration because they can not only integrate exogenous genes efficiently, but also be delivered to a variety of organs using a range of transfection methods. piggyBac (PB) transposon has a high transposability in mammalian cells in vitro, and has been used for genetic and preclinical studies. However, the transposability of PB in mammalian somatic cells in vivo has not been demonstrated yet. Here, we demonstrated PB-mediated sustained gene expression in adult mice. We constructed PB-based plasmid DNA (pDNA) containing reporter [firefly and Gaussia luciferase (Gluc)] genes. Mice were transfected by injection of these pDNAs using a hydrodynamics-based procedure, and the conditions for high-level sustained gene expression were examined. Consequently, gene expressions were sustained over 2 months. Our results suggest that PB is useful for organ-selective somatic integration and sustained gene expression in mammals, and will contribute to basic genetic studies and gene therapies.

γ-Polyglutamic acid-coated vectors for effective and safe gene therapy.

In the present study, we developed some novel gene delivery vectors, coated cationic complexes with gamma-polyglutamic acid (gamma-PGA) for effective and safe gene therapy. Cationic complexes were constructed with pDNA and cationic vectors, such as poly-L-arginine hydrochloride (PLA), poly-L-lysine hydrobromide (PLL), N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA)-cholesterol (Chol) liposomes, and DOTMA-dioleylphosphatidylethanolamine (DOPE) liposomes. The cationic complexes showed high gene expression with strong cytotoxicity in melanoma B16-F10 cells. The cationic complexes were also strongly toxic to erythrocytes. On the other hand, the gamma-PGA was able to coat all cationic complexes and form stable nano-sized particles with negative charges. These gamma-PGA-coated complexes had high gene expression without cytotoxicity and toxicities to the erythrocytes. In in vivo transfection experiments, polyplexes showed high transfection efficiency over 10(5) RLU/g in the lung tissue after intravenous injection, although gamma-PGA-coated polyplexes showed a high value in the spleen. High transfection efficiency in lipoplexes and gamma-PGA-coated lipoplexes was observed in the spleen and lung. Thus, gamma-PGA-coated vectors are useful for clinical gene therapy.