Jun Ohno

A poly(γ-glutamic acid)–amphiphile complex as a novel nanovehicle for drug delivery system

Recently, many studies have focused on biomedical and pharmaceutical applications of self-assembled nanoparticles. In addition, several biodegradable nanoparticles have been reported to possess poor dispersion stability and poor size-controllability. However, these nanoparticles require complicated fabrication procedures using synthesis techniques. We developed an efficient method for producing nanoparticles derived from a biological origin of molecule poly(γ-glutamic acid) (γ-PGA), a cationic lipid, and doxorubicin (Dox). The complex had a size of 510 nm and was able to encapsulate over 90% of the added Dox. An in vivo assay of antitumor activity demonstrated that the complex had significant antitumor activity in sarcoma 180–bearing mice, and was effectively accumulated in solid tumors based on the EPR effect. The data suggested that this complex is a promising formulation of γ-PGA for targeted delivery to solid tumors. γ-PGA–12GP2 complexes may possess several unique advantages, including simplicity of nanoparticle preparation, high drug-carrying capacity, appropriate size to allow deeper penetration based on EPR effect into solid tumors, and lack of necessity to modify the chemical structure of the drugs. These data indicate that the γ-PGA–12GP2 complexes are potentially useful in cancer chemotherapy.

Osteogenic potential for replacing cells in rat cranial defects implanted with a DNA/protamine complex paste.

Osteoinductive scaffolds are required for bone tissue engineering. The aim of the present study was to assess the osteoinductive capacity of deoxyribonucleic acid (DNA)/protamine complexes in a rat model of critical-size calvarial defects. In addition, we investigated whether cultured mesenchymal-like cells (DP-cells) outgrown from DNA/protamine complex engrafted defects could differentiate to become osteogenic cells in vitro. DNA/protamine complexes were prepared by reactions between DNA and protamine sulfate solutions with stirring. Critical-sized (8mm) calvarial defects were created in the central parietal bones of adult rats. Defects were either left empty or treated with DNA/protamine complex scaffolds. Subsequently, micro-computed tomography (micro-CT), histological, and immunohistochemical analyses were performed. Micro-CT and histological assays showed that DNA/protamine complex engrafted defects had enhanced bone regeneration. DP-cells were expanded from explants of DNA/protamine complex engrafted defects using an explant outgrowth culture system. Osteogenesis-related factors were assessed in DP-cells after treatment with an osteoblast-inducing reagent (OIR). After 3months, nearly complete healing was observed for DNA/protamine complex engrafted calvarial defects. Increased alkaline phosphatase (ALP) activity and Alizarin red staining were found for cultured DP-cells. These cells had high expression levels of osteogenic genes, including those for RUNX-2, ALP, osteopontin, and osteocalcin. These results indicated that DNA/protamine complexes could facilitate bone regeneration in calvarial defects. Moreover, in vitro osteogenic induction experiments showed that DP-cells outgrown from DNA/protamine engrafted defects had an osteogenic potential. Based on these results, we suggest that DNA/protamine complexes may recruit osteocompetent cells in these defects, where they differentiate to osteogenic cells.

Preparation and binding study of a complex made of DNA-treated single-walled carbon nanotubes and antibody for specific delivery of a "molecular heater" platform

Carbon nanotubes have been explored as heat-delivery vehicles for thermal ablation of tumors. To use single-walled carbon nanotubes (SWNT) as a “molecular heater” for hyperthermia therapy in cancer, stable dispersibility and smart-delivery potential will be needed, as well as lack of toxicity. This paper reports the preparation of a model complex comprising DNA-treated SWNT and anti-human IgG antibody and the specific binding ability of this model complex with the targeted protein, ie, human IgG. Treatment with double-stranded DNA enabled stable dispersibility of a complex composed of SWNT and the antibody under physiological conditions. Quartz crystal microbalance results suggest that there was one immobilized IgG molecule to every 21,700 carbon atoms in the complex containing DNA-treated SWNT and the antibody. The DNA-SWNT antibody complex showed good selectivity for binding to the targeted protein. Binding analysis revealed that treatment with DNA did not interfere with binding affinity or capacity between the immobilized antibody and the targeted protein. The results of this study demonstrate that the DNA-SWNT antibody complex is a useful tool for use as a smart “molecular heater” platform applicable to various types of antibodies targeting a specific antigen.

Tumor-induced lymphangiogenesis in cervical lymph nodes in oral melanoma-bearing mice

BackgroundMetastasis via the lymphatic system is promoted by lymphangiogenesis. Alterations of the lymphatic channels during the progression of metastasis to regional lymph nodes (LNs) remain unexplored. To examine whether tumor-induced LN lymphangiogenesis controls metastasis to regional LNs, we investigated cervical LN metastasis in a mouse model of oral melanoma.MethodsInjection of B16F10 melanoma cells into mouse tongues replicated spontaneous cervical LN metastasis. We performed histological, immunofluorescent, and histomorphometric analyses of tumor-reactive lymphadenopathy and lymphangiogenesis in tumor-associated LNs. We investigated the expression of vascular endothelial growth factor (VEGF)-C and its receptor, VEGF receptor-3 (VEGFR-3), in tumor cells and tissues, and LNs by reverse transcription polymerase chain reaction and immunofluorescence.ResultsTumor-associated LNs comprised sentinel LNs (SLNs) before and after tumor cell invasion (tumor-bearing SLNs), and LNs adjacent or contralateral to tumor-bearing SLNs. Extensive lymphangiogenesis appeared in SLNs before evidence of metastasis. After metastasis was established in SLNs, both LNs adjacent and contralateral to tumor-bearing SLNs demonstrated lymphangiogenesis. Interaction between VEGF-C-positive melanoma cells and VEGFR-3-positive lymphatic vessels was evident in tumor-associated LNs.ConclusionsLN lymphangiogenesis contributes a progression of tumor metastasis from SLNs to other regional LNs.

Evaluation of bone formation guided by DNA/protamine complex with FGF‐2 in an adult rat calvarial defect model

DNA/protamine complex paste (D/P) and D/P complex paste with Fibroblast Growth Factor-2 (FGF-2) (D/P-FGF) were prepared to investigate their new bone formation abilities using an ∼40-week-old rat calvarial defect model. It was found that D/P could release FGF-2 proportionally in an in vitro experiment with an enzyme-linked immunosorbent assay. It was also found that aging adversely affected self-bone healing of rats by comparison with the results in a previous study using 10-week-old rats. Microcomputed tomography and histopathological examinations showed that new bone formation abilities of D/P and D/P-FGF were superior to that of the control (sham operation). Control, D/P and D/P-FGF showed newly formed bone areas of 6.7, 58.3, and 67.0%, respectively, 3 months after the operation. Moreover, it was found that FGF-2 could support the osteoanagenesis ability of D/P. It was considered that FGF-2 could play an important role in new bone formation at early stages because it induced the genes such as collagen I, CBFA, OSX, and OPN, which are initiated first in the process of osteogenesis. Therefore, D/P-FGF will be a useful injectable biomaterial with biodegradable properties for the repair of bone defects in the elderly.

Cell viabilities and biodegradation rates of DNA/protamine complexes with two different molecular weights of DNA.

Two types of DNA/protamine complexes were prepared from protamine sulfate and 7000 base pair (bp) DNA or original DNA to investigate the effect of the molecular weight of DNA on zeta potential, cell viability, flowability, soft tissue response, and biodegradation rate. The 7000 bp DNA/protamine complex had a negative charge while the original DNA/protamine complex had a positive charge. The cell viabilities (90.4-106.8%) of these complexes were close to each other. The 7000 bp DNA/protamine complex became a softer dough than that of the original DNA/complex when both were kneaded with water. In vivo, the original DNA/protamine complex showed a milder tissue response. The original DNA/protamine complex almost disappeared 30 days after implantation. The 7000 bp DNA/complex disappeared approximately 2 weeks after implantation and areas where samples were implanted became empty. Thereafter, the empty space was gradually replaced by new soft tissues. The original DNA/protamine complex showed low intercalation and groove binding ratios of daunorubicin hydrochloride. Results indicate that high DNA condensation by cationic protamine protected the penetration of degradation enzymes into these complexes. It was found that a high molecular weight of DNA reduced the biodegradation rate and flowability. This study suggests that DNA/protamine complexes could be candidates for biomaterials that control biodegradation rates and flowability.

In vitro and in vivo expression of aldehyde dehydrogenase 1 in oral squamous cell carcinoma.

Aldehyde dehydrogenase isoform 1 (ALDH1) is a useful marker of cancer-initiating cells (CICs) in various organs. In this study, we investigated whether alterations in ALDH1 immunostaining and enzymatic activity in tumor cell populations predicted clinicopathological factors of prognostic importance for cancer progression and contributed to the characteristics of CICs in cisplatin-treated oral squamous cell cancer (OSCC) cells. We evaluated the association between the proportion of ALDH1-positive tumor cells and the clinicopathological features in 90 patients with OSCC. We also examined ALDH1 enzymatic activity, ABCG2 expression, invasive capacity and the ability to self-renew in OSCC cells treated with or without cisplatin. The clinicopathological results showed that elevated ALDH1 expression correlated with local recurrence. In in vitro experiments, the percentage of cells exhibiting ALDH1 enzymatic activity significantly increased among cisplatin-surviving cells (CiSCs) according to flow cytometry. Furthermore, CiSCs demonstrated upregulated expression of ABCG2, their invasive capacity increased, and their ability to generate cancer spheres was enhanced. An increased population of cells exhibiting ALDH1 immunostaining is a predictive marker of local recurrence. ALDH1 expression and activity contributes to the characteristics of CICs in OSCC.

Dynamic changes in cell-surface expression of mannose in the oral epithelium during the development of graft-versus-host disease of the oral mucosa in rats.

BackgroundThe role of cell-surface glycoconjugates in oral mucosal graft-versus-host disease (GVHD) is still unclear, even though molecular changes in the oral epithelium are essential for the pathogenesis of these lesions. In this study, we investigated changes in the binding of mannose (Man)-specific Lens culinaris lectin (LCA) in the oral mucosa of rats with GVHD.MethodsLewis rat spleen cells were injected into (Lewis x Brown Norway) F1 rats to induce systemic GVHD, including oral mucosal lesions. Tongue and spleen samples were evaluated using lectin histochemistry, immunohistochemistry, Western blotting, transwell migration assays and Stamper-Woodruff binding assays.ResultsBinding of Man-specific LCA expanded to the epithelial layers of the tongue in GVHD-rats. An expansion of LCA binding was related to the increased expression of mannosyltransferase in the oral mucosa. CD8+ cells, effector cells of oral mucosal GVHD, expressed mannose-binding protein (MBP) and migrated to the medium containing Man in the transwell migration assay. Adherence of CD8+ cells to the oral epithelium could be inhibited by pretreating CD8+ cells with MBP antibody and/or by pretreating sections with Man-specific LCA.ConclusionsIncreased expression of Man on keratinocytes leads to the migration and/or adhesion of CD8+ cells in the surface epithelium, which is mediated in part by the MBP/Man-binding pathway during the development of oral mucosal GVHD.

Lupus-like oral mucosal lesions in mercury-induced autoimmune response in Brown Norway rats

BackgroundAdministration of mercury at nontoxic doses induces systemic autoimmune disease in Brown Norway (BN) rats. The pathogenesis of lupus-like oral mucosal lesion by mercury-induced autoimmunity is still unclear, even though the oral mucosa is observed to be commonly affected in mercury-treated BN rats. In this study, we investigated the immunopathology of lupus-like oral mucosal lesions in a model of mercury-induced systemic autoimmunity.MethodsBrown Norway male rats were injected subcutaneously with either phosphate-buffered saline (control) or mercury at a dose of 1.0 mg per kilogram of body weight on days 0, 3, 5, and 7. Blood, kidney, and tongue samples were taken at various timepoints for evaluation by immunohistochemistry, RT-PCR, and lupus band test (LBT).ResultsOral mucosal lesions were classified according to three consecutive temporal phases on the basis of infiltration of immunocompetent cells as follows: (phase I) infiltration of MHC class II+ dendritic cells (DC) and macrophages; (phase II) addition of ED1+ macrophage infiltrates; and (phase III) focal infiltration of pan T cells following increased infiltration of DC and macrophages. Dense infiltration of DC and macrophages was observed in the basement membrane (BM) zone of the oral epithelium. Tissue expression of IL-4 mRNA was detected in early lesions (phase I), suggesting that locally produced IL-4 may be responsible for Th2-mediated immune response. A linear and continuous smooth pattern of fluorescence was observed in the oral epithelial BM in addition to renal glomeruli, indicating immune complex deposits.ConclusionsLocal autoimmune responses are involved in the pathogenesis of mercury-induced lupus-like lesions of the oral mucosa.

Salmon DNA Accelerates Bone Regeneration by Inducing Osteoblast Migration

The initial step of bone regeneration requires the migration of osteogenic cells to defective sites. Our previous studies suggest that a salmon DNA-based scaffold can promote the bone regeneration of calvarial defects in rats. We speculate that the salmon DNA may possess osteoinductive properties, including the homing of migrating osteogenic cells. In the present study, we investigated the influence of the salmon DNA on osteoblastic differentiation and induction of osteoblast migration using MG63 cells (human preosteoblasts) in vitro. Moreover, we analyzed the bone regeneration of a critical-sized in vivo calvarial bone defect (CSD) model in rats. The salmon DNA enhanced both mRNA and protein expression of the osteogenesis-related factors, runt-related transcription factor 2 (Runx2), alkaline phosphatase, and osterix (OSX) in the MG63 cells, compared with the cultivation using osteogenic induction medium alone. From the histochemical and immunohistochemical assays using frozen sections of the bone defects from animals that were implanted with DNA disks, many cells were found to express aldehyde dehydrogenase 1, one of the markers for mesenchymal stem cells. In addition, OSX was observed in the replaced connective tissue of the bone defects. These findings indicate that the DNA induced the migration and accumulation of osteogenic cells to the regenerative tissue. Furthermore, an in vitro transwell migration assay showed that the addition of DNA enhanced an induction of osteoblast migration, compared with the medium alone. The implantation of the DNA disks promoted bone regeneration in the CSD of rats, compared with that of collagen disks. These results indicate that the salmon DNA enhanced osteoblastic differentiation and induction of migration, resulting in the facilitation of bone regeneration.