Mai Hazekawa

Prions are misfolded proteins (PrP^Sc) capable of inducing the same conformational change in normal prion proteins (PrP^C). These aberrant proteins are responsible for neurodegenerative diseases in animals and humans, for which no effective treatments currently exist. At present, preventive diagnostic screening remains the only viable intervention. Our research has focused on screening compound libraries to identify small molecules capable of blocking PrP^Sc formation using a prion-infected cell culture model. Over the past several years, we have identified and assembled a set of compounds with diverse chemotypes that inhibit prion activity in human prion-infected cells while maintaining acceptable toxicity profiles, without affecting cell proliferation and morphological change. In this study, we combined the prion-infected cell culture model with surface plasmon resonance (SPR) analysis to screen for novel anti-prion compounds. A previously identified compound set with diverse chemical scaffolds was used as a training set for two-dimensional substructure searches, leading to the discovery of new chemical scaffolds. Representative compounds were selected and evaluated for anti-prion activity, resulting in the identification of two distinct subsets: one that simultaneously suppressed both PrP^Sc and PrP^C levels, and another that selectively inhibited PrP^Sc without affecting PrP^C expression. Correlation analysis between binding affinity parameters (K_d) obtained from SPR and in vitro anti-prion activity demonstrated that concentration-dependent interaction profiles could predict the inhibitory potential of each compound. This integrated approach provides a practical screening strategy to accelerate the elucidation of mechanisms of action and facilitate the discovery of anti-prion agents. All compounds identified in this study are novel and possess favorable chemical features suitable for further structural optimization and rational drug design.

Nucleic acids are highly unstable in vivo due to their physicochemical properties, presenting a major obstacle to their clinical application as therapeutics. Consequently, the development of drug delivery systems (DDS) is essential to enable stable transport of nucleic acids within the body and to promote efficient cellular uptake. In cancer therapy in particular, selective delivery technologies capable of targeting diseased organs or tumor cells are critical for minimizing off-target effects and reducing adverse events.

Exosomes are nano-sized extracellular vesicles that are known to carry various messages to distant cells. It was recently reported that cancer-derived exosomes are orientated to metastatic organs. However, there are no reports on drug carrier development using autologous serum-derived exosomes in vivo. The purpose of this study was to deliver therapeutic siRNAs for melanoma lung metastases using autologous serum-derived exosomes. Primary tumors were induced by subcutaneously injecting melanoma cells into the hindlimbs of female C57BL/6 mice. Primary tumors were surgically removed on day 14. On day 21 after tumor removal, lung metastases were evaluated. Exosomes were isolated from serum collected from mice on days 0, 3, 7, 10, and 14 after primary tumor inoculation. After isolating serum exosomes, siRNA-loaded exosomes were prepared. siRNA-loaded exosomes were intravenously injected into the B16/BL6 spontaneous lung metastasis model mice on days 0, 3, 7, and 10 after tumor removal. siRNA-loaded exosomes prepared with autologous serum-derived exosomes significantly decreased the number of metastatic lung colonies. Autologous serum-derived exosomes, which have high organ accumulation, could potentially be used as efficient carriers of therapeutic siRNAs for melanoma patients with lung metastases.

We previously reported that conjugates of antimicrobial peptide fragment analogues and poly (lactic-co-glycolic) acid (PLGA) enhance antimicrobial activity and that the conjugated micelle structure is an effective tool for antimicrobial drug delivery. In recent years, the delivery of antimicrobial peptides to targets for antimicrobial activity has attracted attention. In this study, we targeted Candida albicans, a causative organism of catheter-related bloodstream infections, which is refractory to antimicrobial agents and is currently a problem in medical practice. We evaluated the antifungal activity of CKR12 (a mutant fragment of the human cathelicidin peptide, LL-37)-PLGA-miconazole (MCZ) micelles using nanotechnology with MCZ delivery. The prepared CKR12-PLGA-MCZ micelles were characterised by measuring dynamic light scattering, zeta potential, dilution stability, and drug release. CKR12-PLGA-MCZ micelles showed higher antifungal activity than CKR12-PLGA micelles and MCZ solution. Furthermore, scanning and transmission electron microscopy suggested that CKR12-PLGA-MCZ micelles disrupted both cell wall and cell membrane of C. albicans. Our results revealed a synergistic effect of antifungal activity using a combination of antimicrobial peptide fragment analogues and MCZ, and that MCZ is a promising tool for the delivery to target microorganisms.

Small interfering RNAs (siRNAs) are susceptible to nucleases and degrade quickly in vivo. Moreover, siRNAs demonstrate poor cellular uptake and cannot cross the cell membrane because of its polyanionic characteristics. To overcome these challenges, an intelligent gene delivery system that protects siRNAs from nucleases and facilitates siRNA cellular uptake is required. We previously reported the potential of siRNA-poly(d,l-lactic-co-glycolic acid; PLGA) micelles as an effective siRNA delivery tool in a murine peritoneal dissemination model by local injection. However, there was no effective formulation for siRNA delivery to target cells via intravenous injection. This study aimed to prepare siRNA–PLGA/Fabʹ–PLGA mixed micelles for siRNA delivery to target floating cells and evaluate its formulation in vitro. As the target siRNA protein in CEMx174, CyclinB1 levels were significantly reduced when siRNA–PLGA/Fabʹ–PLGA mixed micelles were added to cells compared with siRNA–PLGA micelles. siRNA–PLGA/Fabʹ–PLGA mixed micelles have high cell permeability and high target cell accumulation by endocytosis because flow cytometry detected labeling micelles in target cells. This study supports siRNA–PLGA/Fabʹ–PLGA mixed micelles as an effective siRNA delivery tool. This formulation can be administered systemically in dosage form against target cells, including cancer metastasis or blood cancer.

Various peptides and their derivatives have been reported to exhibit antimicrobial activities. Although these activities have been examined against microorganisms, novel methods have recently emerged for conjugation of the biomaterials to improve their activities. Here, we prepared CKR12-PLGA, in which CKR12 (a mutated fragment of human cathelicidin peptide, LL-37) was conjugated with poly (lactic-co-glycolic) acid (PLGA), and compared the antimicrobial and antifungal activities of the conjugated peptide with those of FK13 (a small fragment of LL-37) and CKR12 alone. The prepared CKR12-PLGA was characterized by dynamic light scattering and measurement of the zeta potential, critical micellar concentration, and antimicrobial activities of the fragments and conjugate. Although CKR12 showed higher antibacterial activities than FK13 against Staphylococcus aureus and Escherichia coli, the antifungal activity of CKR12 was lower than that of FK13. CKR12-PLGA showed higher antibacterial activities against S. aureus and E. coli and higher antifungal activity against Candida albicans compared to those of FK13. Additionally, CKR12-PLGA showed no hemolytic activity in erythrocytes, and scanning and transmission electron microscopy suggested that CKR12-PLGA killed and disrupted the surface structure of microbial cells. Conjugation of antimicrobial peptide fragment analogues was a successful approach for obtaining increased microbial activity with minimized cytotoxicity.

Small interfering RNAs (siRNAs) are susceptible to nucleases and degrade quickly in vivo. Moreover, siRNAs demonstrate poor cellular uptake and cannot cross the cell membrane because of its polyanionic characteristics. To overcome these challenges, an intelligent gene delivery system that protects siRNAs from nucleases and facilitates siRNA cellular uptake is required. We previously reported the potential of siRNA-poly(D,L-lactic-co-glycolic acid; PLGA) micelles as an effective siRNA delivery tool in a murine peritoneal dissemination model by local injection. However, there was no effective formulation for siRNA delivery to target cells via intravenous injection. This study aimed to prepare siRNA–PLGA/Fabʹ–PLGA mixed micelles for siRNA delivery to target floating cells and evaluate its formulation in vitro. As the target siRNA protein in CEMx174, CyclinB1 levels were significantly reduced when siRNA–PLGA/Fabʹ–PLGA mixed micelles were added to cells compared with siRNA–PLGA micelles. siRNA–PLGA/Fabʹ–PLGA mixed micelles have high cell permeability and high target cell accumulation by endocytosis because flow cytometry detected labeling micelles in target cells. This study supports siRNA–PLGA/Fabʹ–PLGA mixed micelles as an effective siRNA delivery tool. This formulation can be administered systemically in dosage form against target cells, including cancer metastasis or blood cancer.

Ovarian cancer is known to be the most lethal gynecologic cancer. It has been reported that Glypican-3 (Gpc3) expression induces immune responses, promotes the progression in ovarian cancer. Then, we focused on this Gpc3 gene silencing, tried to prepare siRNA delivery system. In this chapter, we introduce one of the therapeutic proposals in terms of novel drug delivery system using siRNA as a targeting medicine. This chapter introduces our works about preparation of siRNA-PLGA hybrid micelles to deliver the siRNA into the ovarian cancer cells and to evaluate gene silencing effects in mice model. As a result, siRNA-PLGA hybrid micelles were shown to effectively inhibit Gpc3 expression in vitro. In addition, siRNA-PLGA hybrid micelles also decreased the number of tumor nodes in the mesentery in vivo. These results suggested that Gpc3 could be a target molecule for ovarian cancer treatment and siRNA-PLGA hybrid micelles could be an effective siRNA delivery tool even in vivo.

A real‑time cell‑monitoring analysis (RTCA) system was previously developed based on the change in impedance when cells attach and spread in a culture dish coated with a gold microelectrode array. However, the potential applications of this system have not yet been fully demonstrated. The purpose of this study was to test the utility of the RTCA system to determine the cytotoxicity of four anticancer agents in carcinoma cells. The results were compared with those of the conventional WST‑8 assay at the endpoint to determine the potential of the RTCA system as a new real‑time assay method to evaluate cytotoxicity. iCELLigence was used as the RTCA system in this study. Suspensions of oral squamous cell carcinoma (OSCC) cell lines were seeded (2x104 cells/well) onto the E‑plate (the culture plate of the iCELLigence system). After 24 h of culture, anticancer agents were added to each well, and changes in electrical impedance (cell index, CI) were recorded for another 72 h of culture. Cell proliferation was detected in real‑time by the RTCA device in an automated, high throughput manner. Then, the IC50 profiles of the four anticancer agents were calculated based on the real‑time cell index values. The results indicated that the RTCA system was useful in evaluating cytotoxic reactions immediately after the addition of the anticancer agents as it was able to record the data in real‑time. Furthermore, the IC50 levels measured by the real‑time assay were lower than those measured by the endpoint assay. Thus, RTCA systems can be used to evaluate chemotherapeutic agents in cancer cells as well as their side effects in normal cells.