Sayoko Narahara

Development of Human Hepatocellular Carcinoma Cell-Targeted Protein Cages

We described herein a human hepatocellular carcinoma (HCC) cell-targeted protein cage for which the HCC-binding peptide termed SP94 was modified at the surface of a naturally occurred heat shock protein (Hsp) cage. Six types of HCC-targeted Hsp cages were chemically synthesized using two types of heterobifunctional linker (SM(PEG)(n)) with different lengths and two types of SP94 peptide, which contained a unique Cys residue at the N- or C-terminus of the peptide. These Hsp cages were characterized using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analyses, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and dynamic light scattering (DLS) measurement. Fluorescence microscopic observations revealed that all the engineered protein cages bind selectively to HCC cells but not to the other cell lines tested (including normal liver cell). Moreover, the number of SP94 peptides on Hsp cages, conjugation site of SP94 peptide, and linker length between a Hsp cage and a SP94 peptide had important effects upon the binding of engineered Hsp cages to HCC cells. An engineered Hsp cage conjugated to the N-terminus of SP94 peptide via a longer linker molecule and containing high SP94 peptide levels showed greater binding toward HCC cells. Surprisingly, through optimization of these three factors, up to 10-fold greater affinity toward HCC cells was achieved. These results are critically important not only for the development of HCC cell-targeting devices using SP94 peptide, but also to create other cell-targeting materials that utilize other peptide ligands.

Alterations of circadian clockworks during differentiation and apoptosis of rat ovarian cells

Ovarian development is related to cell proliferation, differentiation, and apoptosis of granulosa cells and luteal cells under the control of various modulators, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and growth factors. In the present study, the expression of clock genes and the related regulation mechanism were analyzed in different ovarian cell types during differentiation and apoptosis. The authors focused on the circadian expression of Per2 as a core clock gene for the maintenance of circadian rhythms. By using a real-time monitoring system of the Per2 promoter activity, the circadian oscillation was analyzed in the granulosa and luteal cells from preantral follicles, antral follicles, and corpora lutea of immature Per2 promoter–destabilized luciferase transgenic rats that were primed with diethylstilbestrol, equine chorionic gonadotropin (eCG), and/or human CG. In addition, transcript levels of Per2, Bmal1, Clock, and Nampt were quantified by quantitative polymerase chain reaction (qPCR). Immunohistochemical studies revealed strong circadian rhythmicity of PER2 protein in the luteal cells, but apparently little rhythmicity in granulosa cells of both preantral and antral follicles. In vitro monitoring of promoter activity showed generation of several oscillations in luteal cells after exposure to dexamethasone (DXM), whereas oscillatory amplitudes of immature and mature granulosa cells were rapidly attenuating. The circadian rhythm of the Bmal1 transcript levels, but not the Per2 transcript, was very weak in the granulosa cells, as compared with that in luteal cells. Granulosa cells gained a strong circadian rhythm ability of the Per2 promoter activity after stimulation with FSH for 3 days. In contrast, LH had little effect on the circadian rhythm before stimulation of granulosa cells with FSH, probably owing to lack of LH receptor. In luteal cells, induction of apoptosis by inhibiting progesterone synthesis resulted in deregulation of Per2 circadian oscillation. Transcript levels of Bmal1 and Clock, but not Per2 and Nampt, were significantly decreased in apoptotic luteal cells. The Bmal1 transcript level was particularly reduced. Consequently, these results strongly suggest the circadian clockwork alters in ovarian cells during follicular development, luteinization, and apoptosis, and expression of Bmal1 may be related to the switch-on and switch-off of the circadian oscillation. (Author correspondence: mhattori@agr.kyushu-u.ac.jp)

A nanocarrier based on a genetically engineered protein cage to deliver doxorubicin to human hepatocellular carcinoma cells

Herein, we report the preparation of genetically engineered protein cages (HspG41C-SP94), taken up selectively by human hepatocellular carcinoma (HCC) cells. An engineered protein cage-doxorubicin (DOX) conjugate was as cytotoxic as free DOX against HCC cells but much less cytotoxic against normal hepatocytes.

Liver cell specific targeting by the preS1 domain of hepatitis B virus surface antigen displayed on protein nanocages

Protein nanocages are self-organized complexes of oligomers whose three-dimensional architecture can been determined in detail. These structures possess nanoscale inner cavities into which a variety of molecules, including therapeutic or diagnostic agents, can be encapsulated. These properties yield these particles suitable for a new class of drug delivery carrier, or as a bioimaging reagent that might respond to biochemical signals in many different cellular processes. We report here the design, synthesis, and biological characterization of a hepatocyte-specific nanocage carrying small heat-shock protein. These nanoscale protein cages, with a targeting peptide composed of a preS1 derivative from the hepatitis B virus on their surfaces, were prepared by genetic engineering techniques. PreS1-carrying nanocages showed lower cytotoxicity and significantly higher specificity for human hepatocyte cell lines than other cell lines in vitro. These results suggested that small heat-shock protein-based nanocages present great potential for the development of effective targeted delivery of various agents to specific cells.

Biological evaluation of protein nanocapsules containing doxorubicin

This study describes the applications of a naturally occurring small heat shock protein (Hsp) that forms a cage-like structure to act as a drug carrier. Mutant Hsp cages (HspG41C) were expressed in Escherichia coli by substituting glycine 41 located inside the cage with a cysteine residue to allow conjugation with a fluorophore or a drug. The HspG41C cages were taken up by various cancer cell lines, mainly through clathrin-mediated endocytosis. The cages were detected in acidic organelles (endosomes/lysosomes) for at least 48 hours, but none were detected in the mitochondria or nuclei. To generate HspG41C cages carrying doxorubicin (DOX), an anticancer agent, the HspG41C cages and DOX were conjugated using acid-labile hydrazone linkers. The release of DOX from HspG41C cages was accelerated at pH 5.0, but was negligible at pH 7.2. The cytotoxic effects of HspG41C–DOX against Suit-2 and HepG2 cells were slightly weaker than those of free DOX, but the effects were almost identical in Huh-7 cells. Considering the relatively low release of DOX from HspG41C–DOX, HspG41C–DOX exhibited comparable activity towards HepG2 and Suit-2 cells and slightly stronger cytotoxicity towards Huh-7 cells than free DOX. Hsp cages offer good biocompatibility, are easy to prepare, and are easy to modify; these properties facilitate their use as nanoplatforms in drug delivery systems and in other biomedical applications.

Basic fibroblast growth factor-treated adipose tissue-derived mesenchymal stem cell infusion to ameliorate liver cirrhosis via paracrine hepatocyte growth factor

Recent studies show that adipose tissue‐derived mesenchymal stem cells have potential clinical applications. However, the mechanism has not been fully elucidated yet. Here, we investigated the effect of basic fibroblast growth factor‐treated adipose tissue‐derived mesenchymal stem cells infusion on a liver fibrosis rat model and elucidated the underlying mechanism.

Design and function of engineered protein nanocages as a drug delivery system for targeting pancreatic cancer cells via neuropilin-1

We describe the development of neuropilin 1-binding peptide (iRGD)-nanocages that specifically target human pancreatic cancer cells in which an iRGD is joined to the surface of naturally occurring heat shock protein (HSP) cages. Using a genetic engineering approach, the iRGD domain was joined to the C-terminal region of the HSP cage using flexible linker moieties. The characteristics of the interdomain linkages between the nanocage and the iRGD domain play an important role in the specificity and affinity of the iRGD-nanocages for their target cells. An engineered L30-iRGD-nanocage with 30 amino acid linkers, (GGS)10, showed greater binding affinity for pancreatic cancer cells relative to that of other linkers. Furthermore, a moderately hydrophobic anticancer drug, OSU03012, was successfully incorporated into the L30-iRGD-nanocage by heating the mixture. The OSU03012-loaded L30-iRGD-nanocage induced cell death of pancreatic cancer cells by activating the caspase cascade more effectively than the same concentrations of free OSU03012. The iRGD-nanocages show great potential as a novel nanocarrier for pancreatic cancer-targeted drug delivery.

Systemic Delivery of Protein Nanocages Bearing CTT Peptides for Enhanced Imaging of MMP-2 Expression in Metastatic Tumor Models

Matrix metalloproteinase 2 (MMP-2) in metastatic cancer tissue, which is associated with a poor prognosis, is a potential target for tumor imaging in vivo. Here, we describe a metastatic cancer cell-targeted protein nanocage. An MMP-2-binding peptide, termed CTT peptide (CTTHWGFTLC), was conjugated to the surface of a naturally occurring heat shock protein nanocage by genetic modification. The engineered protein nanocages showed a binding affinity for MMP-2 and selective uptake in cancer cells that highly expressed MMP-2 in vitro. In near-infrared fluorescence imaging, the nanocages showed specific and significant accumulation in tumor tissue after intravenous injection in vivo. These protein nanocages conjugated with CTT peptide could be potentially applied to a noninvasive near-infrared fluorescence detection method for imaging gelatinase activity in metastatic tumors in vivo.

Noninvasive mapping of the redox status of dimethylnitrosamine-induced hepatic fibrosis using in vivo dynamic nuclear polarization-magnetic resonance imaging

Hepatic fibrosis is a chronic disorder caused by viral infection and/or metabolic, genetic and cholestatic disorders. A noninvasive procedure that enables the detection of liver fibrosis based on redox status would be useful for disease identification and monitoring, and the development of treatments. However, an appropriate technique has not been reported. This study describes a novel method for assessing the redox status of the liver using in vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) with the nitroxyl radical carbamoyl-PROXYL as a molecular imaging probe, which was tested in dimethylnitrosamine-treated mice as a model of liver fibrosis. Based on the pharmacokinetics of carbamoyl-PROXYL in control livers, reduction rate mapping was performed in fibrotic livers. Reduction rate maps demonstrated a clear difference between the redox status of control and fibrotic livers according to the expression of antioxidants. These findings indicate that in vivo DNP-MRI with a nitroxyl radical probe enables noninvasive detection of changes in liver redox status.

Splenectomy enhances the therapeutic effect of adipose tissue-derived mesenchymal stem cell infusion on cirrhosis rats

Clinical studies suggest that splenectomy improves liver function in cirrhotic patients, but the influence of splenectomy on stem cell transplantation is poorly understood. This study investigated the effect of splenectomy on stem cell infusion and elucidated its mechanism.