Yoichi Tachibana

Liver-targeted siRNA delivery by polyethylenimine (PEI)-pullulan carrier

Recently, small interfering RNA (siRNA)-based therapeutics have been used to treat diseases. Efficient and stable siRNA delivery into disease cells is important in the use of this agent for treatment. In the present study, pullulan was introduced into polyethylenimine (PEI) for liver targeting. PEI/siRNA or pullulan-containing PEI/siRNA complexes were delivered into mice through the tail vein either by a hydrodynamics- or non-hydrodynamics-based injection. The incidence of mortality was found to increase with an increase in the nitrogen/phosphorus (N/P) ratio of PEI/siRNA complexes. Moreover, the hydrodynamics-based injection increased mice mortality. Introduction of pullulan into PEI dramatically reduced mouse death after systemic injection. After systemic injection, the PEI/fluorescein-labeled siRNA complex increased the level of fluorescence in the lung and the PEI-pullulan/siRNA complex led to an increased fluorescence level in the liver. These results suggest that the PEI-pullulan polymer may be a useful, low toxic means for efficient delivery of siRNA into the liver.

Biodegradable thermoresponsive poly(amino acid)s

Reaction of poly(succinimide) with a mixture of 5-aminopentanol and 6-aminohexanol produced new thermoresponsive polymers based on biodegradable poly(amino acids)s, poly(N-substituted alpha/beta-asparagine)s, showing a clear LCST in water.

The use of magnetic resonance cell tracking to monitor endothelial progenitor cells in a rat hindlimb ischemic model.

A water-soluble magnetic resonance imaging (MRI) contrast agent, Dextran mono-N-succinimidyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate-gadolinium(3+) (Dex-DOTA-Gd(3+)), was shown to enable monitoring of the anatomical migration and the survival period of transplanted stem cells for up to 1 month. Gadolinium molecules in the cells were rapidly eliminated from the site and excreted upon cell death. Endothelial progenitor cells (EPCs) transplanted into the inguinal femoral muscle of rats migrated distally through the knee in rats after hindlimb ischemia but did not migrate in non-ischemic rats. Interestingly, the survival period of transplanted EPCs was notably prolonged in the ischemic limb, indicating that EPCs are required by the ischemic tissues and that the fate of transplanted EPCs was affected by the disease. Compared to the commonly used particle type of MRI contrast agents, the system described in this study is expected to be invaluable to help clarifying the process of stem cell transplantation therapy.

Design and characterization of a polymeric MRI contrast agent based on PVA for in vivo living‐cell tracking

A novel water-soluble MRI contrast agent for in vivo living cell tracking was developed. Unlike the conventional in vivo cell tracking system based on superparamagnetic iron oxide beads, the newly developed contrast agent is eliminated from the body when the contrast agent exits the cells upon cell death, which makes living cell tracking possible. The contrast agent is composed of gadolinium chelates (Gd-DOTA) and a water-soluble carrier, poly(vinyl alcohol) (PVA), which is known to interact with cells and tissues very weakly. Since the Gd-PVA was not taken up by cells spontaneously, the electroporation method was used for cell labeling. The delivered Gd-PVA was localized only in the cytosolic compartment of growing cells with low cytotoxicity and did not leak out of the living cells for long periods of time. This stability may be due to the weak cell-membrane affinity of Gd-PVA, and did not affect cell proliferation at all. After cell labeling, signal enhancement of cells was observed in vitro and in vivo. These results indicate that Gd-PVA can visualize only the living cells in vivo for a long period of time, even in areas deep within large animal bodies.

Long-Term/Bioinert Labeling of Rat Mesenchymal Stem Cells with PVA-Gd Conjugates and MRI Monitoring of the Labeled Cell Survival after Intramuscular Transplantation

Noninvasive in vivo imaging of transplanted stem cells is an effective method to clarify the mechanisms involved in stem cell transplantation therapy. We labeled rat mesenchymal stem cells (MSCs) with water-soluble magnetic resonance imaging (MRI) contrast agent poly(vinyl alcohol)-gadolinium (PVA-Gd) in order to ascertain the fate of transplanted MSCs in vivo. PVA-Gd was retained and localized in the cytosolic compartment of MSCs for a longer period of time. The effect of PVA-Gd labeling on MSC proliferation was much less than that of the commercially available contrast agent ProHance, and the labeled MSCs were found to have osteoblastic differentiation ability. To study the MSC lifetime in vivo, MSCs were seeded and trapped in the cytocompatible three-dimensional porous scaffolds of Spongel and transplanted. The MRI signal attributed to MSCs was eliminated from the transplanted site in 14 days. Because free PVA-Gd was rapidly eliminated from the site, this signal reduction indicated MSC death in the transplantation site. The low efficiency of MSC transplantation for ischemic tissue may be due to their short lifetime, making it important to develop highly effective stem cell transplantation systems that address cell number, injection position, and cell formulation (suspension, sheet, and aggregates). Our cell survival tracking system would be a very powerful tool to this end and would be applicable in clinical cell therapies.

Efficient reduction of serum cholesterol by combining a liver-targeted gene delivery system with chemically modified apolipoprotein B siRNA

Apolipoprotein B (Apo B) is a key amphipathic glycoprotein compound in the metabolism of plasma lipoproteins (mainly very low-density lipoprotein (VLDL) and LDL). Inhibition of Apo B synthesis by short interfering RNA (siRNA) targeting Apo B (Apo B siRNA) is very efficient for serum LDL reduction. In the present study, the chemically modified Apo B siRNA (Apo B-siBNA) with the increased enzymatic stability was selected. We developed a cationic conjugate for efficient delivery of Apo B-siBNA into the liver by introducing pullulan with different molecular weights (MWs) (5900 and 107,000) into polyethylenimine (PEI). Introduction of pullulan into PEI dramatically decreased mortality and lung damage after systemic injection of the conjugate/Apo B-siBNA complexes into mice. The PEI-pullulan carrier prepared with high MW pullulan (107,000) was more stable in the blood stream and showed higher fluorescence levels in the liver for a longer time than the carrier prepared with low MW pullulan (5900). Moreover, efficient reduction of serum LDL and Apo B mRNA in the liver was observed in mice injected with PEI-pullulan (MW, 107,000)/Apo B-siBNA, whereas there was no or little change in serum LDL and Apo B mRNA in livers of mice treated with Apo B-siBNA alone, PEI/Apo B-siBNA, and PEI-pullulan (MW, 5900)/Apo B-siBNA. These results suggest that combining a liver-targeted gene delivery system with chemically modified Apo B siRNA efficiently reduces the level of serum LDL and Apo B mRNA in the liver.

Quick nuclear transportation of siRNA and in vivo hepatic ApoB gene silencing with galactose-bearing polymeric carrier.

Since previous studies have linked the genetic mutations of Apolipoprotein B (ApoB) to the low density lipoprotein (LDL) cholesterol levels, it can be believed that the knockdown of ApoB by siRNA silencing is a useful method to reduce the cardiovascular disease. However, the spontaneous uptake of siRNA is hindered, and thus vectors are necessary to aid its transfer into the cells. Among the synthetic non-viral vectors, cationic polymers are extensively investigated as possible candidates for efficient and specific gene delivery, because they can be easily modified to get different set of properties. Therefore, in this work a set of random copolymers with different molecular weight and composition were synthesized. These vectors present 2-(dimethylamino)ethyl methacrylate, as cationic monomer, and galactose units as liver-targeting moieties. From in vitro experiments, copolymers with monomer ratio and molecular weight about 0.1 and 80kDa, respectively, showed adequate transfection capabilities and displaying good cell viability, independently of the nature of the saccharides units. However, in the in vivo experiments in C57BL/6 high-fat-fed mice, a better blood compatibility and protection against degradation leading to better transfection by the random copolymers bearing galactose units was confirmed.

Hepatocyte-Specific Gene Delivery with Galactose-Bearing Cationic Polymers with Different Molecular Structures

Since the promising virus -based gene therapies are often limited by problems such as the immunity of virus itself, the development of an efficient non-viral vector is of prime importance. For this reason, several synthetic nonviral polymeric carriers including cationic sequences have been molecularly designed. It is well known that the polymeric carriers with some cationic groups buffer the endosomal pH resulting in the enhanced transfection efficiency, but also in a relatively high toxicity. In the last decades, the polymers bearing pendant carbohydrates (glycopolymers) was proved to have relatively less toxic. Since the glycopolymers may not only decrease the toxicity of the cationic chain but also serve as targeting agent, we have rationally designed new glycopolymer-based gene delivery carriers. The interaction of carrier/gene polyplexes with hepatocytes and their intracellular trafficking were investigated in vitro. Our results show the significant efficacy of the galactose moieties on the uptake by hepatocytes, in a ligand specific manner.

Synthesis, properties, and endothelial progenitor cells labeling stability of dextranes as polymeric magnetic resonance imaging contrast agents

Magnetic resonance imaging is one of the most important fields for cellular imaging due to its non-invasive capacity, spatial resolution, and sensibility to visualized transplanted cells. An enhanced magnetic resonance image can be achieved by using contrast agents containing paramagnetic gadolinium chelates, which have the widest clinical use. To obtain a better contrast-enhancement and reduce the concentration of Gd for payload, one strategy is to conjugate the gadolinium(III) chelate to polymeric materials that will lead into an increase in the rotational correlation time and therefore improve the relaxivity. Four series of dextran gadolinium chelates were synthesized which are of interest as potential MRI contrast agents to track bone marrow-derived endothelial progenitor cells in vivo. The dextranes with molecular weights were characterized, introduced into the endothelial progenitor cells by electroporation, and injected in aqueous solution into rats to acquire the MR images. We have shown that by selecting polymers of the appropriate molecular weight, stability into the cell after labeling, relaxivity, and retention into the body can be accomplished.