Yuriko Higuchi

Luminescent proteins for high-speed single-cell and whole-body imaging

The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca2+, cyclic adenosine monophosphate and adenosine 5′-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.

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.

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.

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.

Systemic Targeting of Lymph Node Metastasis through the Blood Vascular System by Using Size-Controlled Nanocarriers.

Occult nodal metastases increase the risk of cancer recurrence, demoting prognosis and quality of life of patients. While targeted drug delivery by using systemically administered nanocarriers can potentially control metastatic disease, lymph node metastases have been mainly dealt by locally injecting nanocarriers, which may not always be applicable. Herein, we demonstrated that sub-50 nm polymeric micelles incorporating platinum anticancer drugs could target lymph node metastases in a syngeneic melanoma model after systemic injection, even after removing the primary tumors, limiting the growth of the metastases. By comparing these micelles with clinically used doxorubicin-loaded liposomes (Doxil) having 80 nm, as well as a 70 nm version of the micelles, we found that the targeting efficiency of the nanocarriers against lymph node metastases was associated with their size-regulated abilities to extravasate from the blood vasculature in metastases and to penetrate within the metastatic mass. These findings indicate the potential of sub-50 nm polymeric micelles for developing effective conservative treatments against lymph node metastasis capable of reducing relapse and improving survival.

Photothermic regulation of gene expression triggered by laser-induced carbon nanohorns

The development of optical methods to control cellular functions is important for various biological applications. In particular, heat shock promoter-mediated gene expression systems by laser light are attractive targets for controlling cellular functions. However, previous approaches have considerable technical limitations related to their use of UV, short-wavelength visible (vis), and infrared (IR) laser light, which have poor penetration into biological tissue. Biological tissue is relatively transparent to light inside the diagnostic window at wavelengths of 650–1,100 nm. Here we present a unique optical biotechnological method using carbon nanohorn (CNH) that transforms energy from diagnostic window laser light to heat to control the expression of various genes. We report that with this method, laser irradiation within the diagnostic window resulted in effective heat generation and thus caused heat shock promoter-mediated gene expression. This study provides an important step forward in the development of light-manipulated gene expression technologies.

Enhanced Anti-Inflammation of Inhaled Dexamethasone Palmitate Using Mannosylated Liposomes in an Endotoxin-Induced Lung Inflammation Model

Inhalation of bacterial endotoxin induces pulmonary inflammation by activation of nuclear factor κB (NFκB), production of cytokines and chemokines, and neutrophil activation. Although glucocorticoids are the drugs of choice, administration of free drugs results in adverse effects as a result of a lack of selectivity for the inflammatory effector cells. Because alveolar macrophages play a key role in the inflammatory response in the lung, selective targeting of glucocorticoids to alveolar macrophages offers efficacious pharmacological intervention with minimal side effects. We have demonstrated previously the selective targeting of mannosylated liposomes to alveolar macrophages via mannose receptor-mediated endocytosis after intratracheal administration. In this study, the anti-inflammatory effects of dexamethasone palmitate incorporated in mannosylated liposomes (DPML) at 0.5 mg/kg via intratracheal administration were investigated in lipopolysaccharide-induced lung inflammation in rats. DPML significantly inhibited tumor necrosis factor α, interleukin-1β, and cytokine-induced neutrophil chemoattractant-1 levels, suppressed neutrophil infiltration and myeloperoxidase activity, and inhibited NFκB and p38 mitogen-activated protein kinase activation in the lung. These results prove the value of inhaled mannosylated liposomes as powerful targeting systems for the delivery of anti-inflammatory drugs to alveolar macrophages to improve their efficacy against lung inflammation.

Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages.

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.

Development of lysine–histidine dendron modified chitosan for improving transfection efficiency in HEK293 cells

Chitosan has potential as a biocompatible gene carrier. However, its gene transfection efficiency is low because of its slow endosomal escape rate. Histidine has buffering capacity in the pH range of endosomes/lysosomes. The structure of dendron consists of a central core with several chains radiating from it and many histidines could be conjugated on the surface, increasing the efficiency of histidine modification. The purpose of this study is to increase the gene transfection efficiency of chitosan by promoting its endosomal escape property. We developed fourth-generation lysine-histidine (KH) dendrons that can provide 8 histidines in one dendron molecule. Chitosan-dendron (Chi13k-D) was synthesized using 2-iminothiolane to form the linkage; this was confirmed by NMR and the ninhydrin test. The buffering range, as measured by pH titration, was broader in the Chi13k-D group than in chitosan. Enhanced endosomal escape of Chi13k-D/pDNA complexes was confirmed using fluorescence-labeled endosomes and pDNA. The intralysosomal pH of Chi13k-D/pDNA was also higher than that of chitosan/pDNA. The gene transfection efficiency of Chi13k-D/pDNA was higher than that of chitosan/pDNA in HEK293 cells. These results suggest that KH dendron modification could provide high buffering capacity, which would increase the gene transfection efficiency of chitosan.