Toshihiro Akaike

Preparation of alginate/galactosylated chitosan scaffold for hepatocyte attachment.

Galactose-carrying lactobionic acid was covalently coupled with chitosan for determining hepatocyte specificity. Galactosylated chitosan (GC) was reacted with Ca-alginate (ALG) gel through the electrostatic interaction of carboxylic groups of alginate with amine groups of GC. Highly porous, three-dimensional sponge composed of ALG and GC was prepared to provide specific hepatocyte recognition signals and enhance the mechanical property of the ALG sponge. Observation of the sponge through scanning electron microscopy revealed that sponge was a highly porous microstructure with interconnected pores. Porosity and pore size of the sponge were greatly dependent on the content and molecular weight of GC, and freezing temperature. The mechanical property of the ALG/GC sponge was enhanced with an increase of the GC content. Spheroid formation and viability of hepatocytes of the ALG/GC sponge were higher than those of the ALG one.

Galactosylated alginate as a scaffold for hepatocytes entrapment.

Galactose moieties were covalently coupled with alginate through ethylenediamine as the spacer for enhancing the interaction of hepatocytes with alginate. Adhesion of hepatocytes onto the galactosylated alginate (GA)-coated polystyrene (PS) surface showed an 18-fold increase as compared with that of the alginate-coated surface and it increased with an increase in the concentration of GA. The morphologies of attached hepatocytes were observed to spread out at the 0.15 wt% GA-coated PS surface while round cells were observed at the 0.5 wt% GA-coated PS surface. Inhibition of hepatocytes attachment onto the galactose-carrying PS-coated surface occurred with the addition of the GA into the hepatocyte suspension, indicating the binding of GA with hepatocytes via the patch of asialoglycoprotein receptors. Primary hepatocytes were entrapped in the GA/Ca2+ capsules (GAC). Higher cell viability and more spheroid formation of hepatocytes were obtained in the GAC than in the alginate/Ca2+ capsules (AC). Moreover, liver functions of the hepatocytes such as albumin secretion and urea synthesis in the GAC were improved in comparison with those in the AC.

A new non-viral DNA delivery vector: the terplex system

A new DNA delivery vector (the terplex system) based on a balanced hydrophobicity and net surface charge between stearyl-poly(L-lysine), low density lipoprotein (LDL), and genetic material (i.e. plasmid DNA or antisense oligonucleotide) was developed. The pSV-beta-gal plasmid in terplex system showed a 2-5-fold increase in beta-galactosidase expression on murine smooth muscle cells (A7R5) compared to Lipofectin. Delivery of unmodified c-myb antisense oligonucleotide to A7R5 cells was also facilitated significantly by the terplex system, requiring as little as 5.4 nM of antisense oligonucleotide to achieve a 50% antiproliferative effect. Similar antiproliferative effect was observed when the c-myb antisense/terplex formulation was tested on CCD-32 Lu human lung fibroblasts. Characterization of the physical properties of the terplex system was performed using various techniques. Plasmid DNA was condensed by addition of stearyl-PLL and LDL, resulting in the terplex system of about 100 nm in diameter as shown by atomic force microscopy. A strong hydrophobic interaction between stearyl-poly(L-lysine) and LDL was registered by 1H-NMR spectrometry, showing a significant decrease in the epsilon-methylene signal of poly(L-lysine) backbone when stearyl-poly(L-lysine) was mixed with LDL; however, this phenomenon was not observed with unmodified poly(L-lysine). Agarose gel electrophoresis revealed that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-poly(L-lysine), indicating that the surface charge of the terplex system became more positive by addition of stearyl-poly(L-lysine). Zeta-potential measurement showed that the terplex system exerted a slightly positive charge (+2 mV) at a 1:1:1 weight ratio of plasmid DNA:LDL:stearyl-poly(L-lysine). The obtained results will be utilized in the design of more efficient and safer DNA delivery vectors for in vivo gene therapy.

Hepatocyte-specific porous polymer-scaffolds of alginate/galactosylated chitosan sponge for liver-tissue engineering

Porous scaffolds of alginate/galactosylated chitosan (ALG/GC) sponges were prepared by lyophilization for liver-tissue engineering. Primary hepatocytes in ALG/GC sponges showed higher cell attachment and viability than in alginate alone owing to the specific interaction of the asialoglycoprotein receptors on hepatocyte with the galactose residues on ALG/GC sponges. Improvements in spheroid formation and long-term liver-specific functions of the immobilized hepatocyte were also observed in ALG/GC sponge.

Receptor-mediated delivery of all trans-retinoic acid to hepatocyte using poly(l-lactic acid) nanoparticles coated with galactose-carrying polystyrene

All trans-retinoic acid (RA)-loaded poly(L-lactic acid) (PLA) nanoparticles coated with galactose-carrying polymer, as hepatocyte-specific targeting material using galactose ligands as recognition signals to asialoglycoprotein receptors were prepared by the diafiltration method. Effects of released RA from its loaded nanoparticles on morphology and DNA synthesis of hepatocytes were studied. Receptor-mediated endocytosis of the nanoparticles was checked by fluorescence and confocal laser microscopy. It was found that the shapes of most hepatocytes attached onto polystyrene dish precoated with collagen solution were flat and spreading at low concentration of RA for the RA-loaded nanoparticles, whereas their shapes were round at even low concentration of RA when RA was mixed with the nanoparticles. From the fluorescence and confocal laser microscopic studies, it was suggested that the nanoparticles coated with galactose-carrying polymers were internalized by the hepatocytes through the receptor-mediated mechanism. The RA-loaded nanoparticles were more potent stimulators of hepatocyte DNA synthesis than the free RA system in the presence of epidermal growth factor (EGF) owing to the controlled release of RA from the RA-loaded nanoparticles.

Receptor-mediated cell modulator delivery to hepatocyte using nanoparticles coated with carbohydrate-carrying polymers

Cell modulators such as colchicine (CO), cytochalasin B (CY) and taxol (TX) loaded nanoparticles coated with carbohydrate-carrying polymers, as hepatocyte-specific targeting material using galactose ligands as recognition signals to asialoglycoprotein receptors were prepared by the diafiltration method. Effects of cell modulators from their loaded nanoparticles on morphology of hepatocytes were studied. Receptor-mediated endocytosis of the nanoparticles were examined by fluorescence and confocal laser microscopy. It was found that the shapes of most hepatocytes were changed for the CY-loaded, TX-loaded, or CO-loaded nanoparticles whereas their shapes were not changed in comparison with control when CY, TX, or CO were mixed with the nanoparticles. From the fluorescence and confocal laser microscopic studies, it is suggested that the nanoparticles coated with sugar-carrying polymers were internalized by the hepatocytes through the receptor-mediated mechanism.

Gd3+-loaded polyion complex for pH depiction with magnetic resonance imaging

In clinical diagnosis, gadolinium (Gd) ion/low molecular weight chelater complexes have been used as MRI contrast agents that disperse throughout a particular tissue and cause a brighter appearance in MRI. In order to provide a novel imaging concept for MRI, a contrast agent in which the T(1)-relaxation shortening activity (R(1) relaxivity) changes in response to the pH differences was studied. We prepared a polyion complex (PIC) consisting of a polyanionic Gd-chelater, poly(diethylenetriamine-N,N,N,N, N-pentaaceto, DTPA) (1,3-propanediamide) (denoted as 1a) loaded with Gd ions at a [Gd]/[DTPA unit] ratio of 0.2 (denoted as 1b), and a polycation, poly[2-(diethylamino)ethyl methacrylate] (denoted as 2). The stoichiometric (based on ionic groups) mixture of 1b and 2 formed complex coacervates from pH 5 to pH 8. The R(1) relaxivity of Gd(3+) in the complex was considerably influenced by the pH, and the relative signal intensity changed from 4 at pH 7.2 to 11 at pH 5.0, as determined by an MRI phantom study. The pH responsivity of the complex solution varied with the composition of the PIC (i.e., the mixing ratio of 1b and 2), allowing us to modulate the pH sensibility. The ionic charge balance and swelling of PIC seemingly were related to the pH-dependent R(1) relaxivity change. It is expected that the PIC-based MRI contrast agent may provide a novel category of MRI methods and be useful in improving the detectability of an MRI-based diagnosis.

Possible involvement of caspase-like family in maintenance of cytoskeleton integrity.

Caspases, a family of cysteine proteases, are the key effector proteins of apoptosis. These proteases cleave cellular proteins and are responsible for the destruction of the cell body during apoptosis. They are also involved in the activation of other proteins, such as cytokines. In this study, we demonstrate a novel function for these proteases. Z‐Asp‐CH2‐DCB (Z‐Asp), a general caspase inhibitor, blocked cell spreading on collagen‐coated plates in a dose‐dependent manner but did not affect cell viability. Caspase 3–like activity but not caspase 1–like activity was detected in adherent cells on both collagen‐coated and poly‐L‐lysine–coated plates but not in suspended cells. The caspase 3–like activity was significantly inhibited by Z‐Asp. However, only Z‐Asp, not specific caspase inhibitors (Z‐DEVD for caspase 3, Z‐YVAD for caspase 1), was effective in the suppression of cell spreading. The inhibitory effect of Z‐Asp was blocked by a phosphokinase C activator, PMA, and a Rho activator, lysophosphatidic acid (LPA), while neither a Rac activator, bradykinin, nor a Cdc42 activator, sphingosine‐1‐phosphate, was effective. Immunoprecipitation demonstrated that Z‐Asp downregulated the expression of focal adhesion kinase (FAK) protein, downstream of Rho signaling, in adherent cells. Our results suggest that not caspase 1 or 3 but another yet unknown caspase(s) plays an important role in the maintenance of cytoskeleton integrity via FAK protein expression, implying a new function for caspases. J. Cell. Physiol. 179:45–51, 1999.

Specific adhesion of primary hepatocytes to a novel glucose-carrying polymer

A new amphiphilic glycopolymer, poly-[N-p-vinylbenzyl-d-glucuronamide] (PV6Gna), was synthesized and characterized. Glucose moieties in the polymer were confirmed to be exposed into outer surface of polystyrene (PS) by direct lectin-enzyme assay. Hepatocytes were specifically interacted with PV6Gna substituted at C-6 of glucose but not poly-[N-p-vinylbenzyl-O-α-d-glucopyranosyl-[1→4]-d-gluconamide] (PVMA) and poly-[3-O-p-vinylbenzyl-d-glucose (PVG) substituted at C-1 and C-3 of glucose, respectively, although the glycopolymers were interacted with Con A as lectin for α-d-glucose and α-d-mannose. The adhesion of hepatocytes was dependent on Ca2+ and independent on temperature for the PV6Gna surface unlike integrin-dependent adhesion. Morphologies of hepatocytes on the PV6Gna surface were significantly different from ones on collagen type-I and affected by the coating concentration of PV6Gna onto PS dish and epidermal growth factor (EGF).

Establishment of heterotropic liver tissue mass with direct link to the host liver following implantation of hepatocytes transfected with vascular endothelial growth factor gene in mice.

One of the major goals of tissue engineering is to establish an integrated organ in vivo. We have previously shown that transfection of vascular endothelial growth factor (VEGF) gene into hepatocytes promotes tissue formation by engrafted cells. Here we show that tissue growth was significantly enhanced by co-transplantation of hepatocyte growth factor (HGF) and tumor necrosis factor-alpha (TNF alpha) gene transfected hepatocytes with VEGF-gene transfected cells, but tissue islands were scattered nonspecifically in the abdomen of mice. The result brought us forward to the next step to establish an integrated mass and structural formation of liver tissue. We entrapped VEGF gene transfected hepatocytes in a nylon mesh bag and intraperitoneally engrafted close to the liver. Three weeks later, the bag was covered by a thick network of blood vessels, compared to the control. Histological examination showed that the blood vessels penetrated the parenchyma of the engrafted bag and formed a well-developed vessel network in the region. The use of hepatocytes from lacZ transgenic mice and PCR analysis demonstrated survival and albumin production by hepatocytes in the engrafted bag. Our model can potentially be developed into a heterotropic artificial liver with direct access to the host blood circulation.