Yasutaka Mori

Antiviral activity of silver nanoparticle/chitosan composites against H1N1 influenza A virus

Silver nanoparticle (Ag NP)/chitosan (Ch) composites with antiviral activity against H1N1 influenza A virus were prepared. The Ag NP/Ch composites were obtained as yellow or brown floc-like powders following reaction at room temperature in aqueous medium. Ag NPs (3.5, 6.5, and 12.9 nm average diameters) were embedded into the chitosan matrix without aggregation or size alternation. The antiviral activity of the Ag NP/Ch composites was evaluated by comparing the TCID50 ratio of viral suspensions treated with the composites to untreated suspensions. For all sizes of Ag NPs tested, antiviral activity against H1N1 influenza A virus increased as the concentration of Ag NPs increased; chitosan alone exhibited no antiviral activity. Size dependence of the Ag NPs on antiviral activity was also observed: antiviral activity was generally stronger with smaller Ag NPs in the composites. These results indicate that Ag NP/Ch composites interacting with viruses exhibit antiviral activity.

Preparation and characterization of low-molecular-weight heparin/protamine nanoparticles (LMW-H/P NPs) as FGF-2 carrier

We produced low-molecular-weight heparin/protamine nanoparticles (LMW-H/P NPs) as a carrier for heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2). A mixture of low-molecular-weight heparin (MW: about 5000 Da, 6.4 mg/mL) and protamine (MW: about 3000 Da, 10 mg/mL) at a ratio of 7:3 (vol:vol) yields a dispersion of microparticles (1–6 μm in diameter). In this study, diluted low-molecular-weight heparin solution in saline (0.32 mg/mL) mixed with diluted protamine (0.5 mg/mL) at a ratio at 7:3 (vol:vol) resulted in soluble nanoparticles (112.5 ± 46.1 nm in diameter). The generated NPs could be then stabilized by adding 2 mg/mL dextran (MW: 178–217 kDa) and remained soluble after lyophilization of dialyzed LMW-H/P NP solution. We then evaluated the capacity of LMW-H/P NPs to protect activity of FGF-2. Interaction between FGF-2 and LMW-H/P NPs substantially prolonged the biological half-life of FGF-2. Furthermore, FGF-2 molecules were protected from inactivation by heat and proteolysis in the presence of LMW-H/P NPs.

Preparation of Size-Controlled Silver Nanoparticles and Chitin-Based Composites and Their Antimicrobial Activities

A simple method for the preparation of size-controlled spherical silver nanoparticles (Ag NPs) was reported for their generation by autoclaving a mixture of silver-containing glass powder and glucose. The particle size is regulated by the glucose concentration, with concentrations of 0.25, 1.0, and 4.0 wt% glucose providing small ( nm in diameter), medium ( nm), and large ( nm) particles, respectively. In this study, Ag NP/chitin composites were synthesized by mixing each of these three Ag NP suspensions with a <5% deacetylated (DAc) chitin powder (pH 7.0) at room temperature. The Ag NPs were homogenously dispersed and stably adsorbed onto the chitin. The Ag NP/chitin composites were obtained as yellow or brown powders. Approximately 5, 15, and 20 μg of the small, medium, and large Ag NPs, respectively, were estimated to maximally adsorb onto 1 mg of chitin. The bactericidal and antifungal activities of the Ag NP/chitin composites increased as the amount of Ag NPs in the chitin increased. Furthermore, smaller Ag NPs (per weight) in the chitin composites provided higher bactericidal and anti-fungal activities.

Fragmin/Protamine Microparticle‐Coated Matrix Immobilized Cytokines to Stimulate Various Cell Proliferations With Low Serum Media

Fragmin/protamine microparticles (F/P MPs) have been shown to bind to culture plates, thereby retaining heparin-binding cytokines. Most protocols for in vitro cultures of human microvascular endothelial cells (hMVECs), human dermal fibroblast cells (hDFCs), and hematopoietic cell line (TF-1) include high fetal bovine serum (FBS) (10%) medium as a nutritional supplement. Growth rates of those cells on the F/P MP-coated plates were higher in low FBS (1%) medium containing fibroblast growth factor (FGF)-2 (for hMVECs and hDFCs) and interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor (for TF-1 cells) than without coating. The cytokines in low FBS medium were shown to be immobilized on the F/P MP-coated plate and released into the culture medium with a half releasing time of 4-5 days. Furthermore, those cells grew well on each cytokine-preimmobilized F/P MP-coated plate in low FBS medium. Thus, the F/P MP-coated matrix with adequate heparin-binding cytokines may provide biomaterials for controlling cellular growth and differentiation.

Adsorption of Silver Nanoparticles onto Different Surface Structures of Chitin/Chitosan and Correlations with Antimicrobial Activities

Size-controlled spherical silver nanoparticles (Ag NPs) can be simply prepared by autoclaving mixtures of glass powder containing silver with glucose. Moreover, chitins with varying degrees of deacetylation (DDAc < 30%) and chitosan powders and sheets (DDAc > 75%) with varying surface structure properties have been evaluated as Ag NP carriers. Chitin/chitosan-Ag NP composites in powder or sheet form were prepared by mixing Ag NP suspensions with each of the chitin/chitosan-based material at pH 7.3, leading to homogenous dispersion and stable adsorption of Ag NPs onto chitin carriers with nanoscale fiber-like surface structures, and chitosan carriers with nanoscale porous surface structures. Although these chitins exhibited mild antiviral, bactericidal, and antifungal activities, chitin powders with flat/smooth film-like surface structures had limited antimicrobial activities and Ag NP adsorption. The antimicrobial activities of chitin/chitosan-Ag NP composites increased with increasing amounts of adsorbed Ag NPs, suggesting that the surface structures of chitin/chitosan carriers strongly influence adsorption of Ag NPs and antimicrobial activities. These observations indicate that chitin/chitosan-Ag NPs with nanoscale surface structures have potential as antimicrobial biomaterials and anti-infectious wound dressings.

Fragmin/protamine microparticles as cell carriers to enhance viability of adipose-derived stromal cells and their subsequent effect on in vivo neovascularization.

We prepared fragmin/protamine microparticles (F/P MPs) as cell carriers to enhance cell viability. Use of material consisting of a low-molecular-weight heparin (fragmin) mixed with protamine resulted in water-insoluble microparticles (about 0.5-1 microm in diameter). In this study, we investigated the capability of F/P MPs to enhance the viabilities of human microvascular endothelial cells (HMVECs), human dermal fibroblasts (fibroblasts), and adipose tissue-derived stromal cells (ATSCs) in suspension culture. F/P MPs were bound to the surfaces of these cells, and the interaction of these cells with F/P MPs induced cells/F/P MPs-aggregate formations in vitro, and maintained viabilities of those cells for at least 3 days. The ATSCs/F/P MPs-aggregates adhered to and grew on suspension culture plates in a fashion similar to those on type I collagen-coated plates. The cultured ATSCs secreted significant amounts of angiogenic heparin-binding growth factors such as FGF-2. When the ATSCs/F/P MPs-aggregates were subcutaneously injected into the back of nude mice, significant neovascularization and fibrous tissue formation were induced near the site of injection from day 3 to week 2. The ATSCs/F/P MPs-aggregates were thus useful and convenient biomaterials for cell-therapy of angiogenesis.

Human Stem Cell Factor (SCF) is a Heparin-Binding Cytokine

Binding affinities of chemically modified heparins for human stem cell factor (SCF) were examined using fragmin/protamine microparticles (F/P MPs) and an enzyme-linked immunosorbent assay (ELISA). The binding of SCF to F/P MP-coated plates was inhibited with high concentrations of heparin and fragmin, but not others. The binding of SCF was also inhibited with 0.55 M or higher concentrations of NaCl in the medium. These results suggested that a high content of all three sulfate groups in repeating disaccharide units is required for interaction with SCF. Furthermore, pre-immobilized SCF on F/P MP-coated plates significantly stimulated proliferation of a human erythroleukemia cell line.

Effective expansion of human adipose-derived stromal cells and bone marrow-derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles-coated substratum with human platelet-rich plasma.

Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet‐rich plasma (PRP). In this study, we investigated the capability of F/P NP‐coated plates to act as a substratum for the proliferation of human adipose‐derived stromal cells (ASCs) and bone marrow‐derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP‐coated plates and grew optimally, with a doubling time of 30 and 32 h in low‐concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor‐2 (FGF‐2) on the F/P NP‐coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF‐2 on F/P NP‐coated plates pretreated with PRP and FGF‐2 in a concentration‐dependent manner. Thus, F/P NP‐coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low‐concentration PRP medium supplemented with FGF‐2. No xenogeneic serum is required. Copyright

Increased survival of free fat grafts and vascularization in rats with local delivery of fragmin/protamine microparticles containing FGF-2 (F/P MP-F)

We evaluated the effects of fragmin/protamine micro-particles (F/P MPs) containing FGF-2 (F/P MP-F) as carriers for the controlled release of FGF-2 for adipocyte-survival and capillary formation in inbred rats with subdivided free fat grafts. F/P MPs could immobilize FGF-2, thereafter gradually releasing the bound FGF-2. Inbred Fisher 344 rats weighing around 150 g were anesthetized and implanted with paste comprising harvested fat combined with F/P MP-F. The effect of F/P MP-F on the survival, granulation, and capillary formation in fat grafts was histologically compared with control grafts containing either FGF-2, F/P MPs or PBS. The control fat grafts became attached to tissues adjacent to the implantation site and were significantly resorbed after 30 days. In contrast, pink, soft, supple grafts were compressible and were little resorbed in the group given F/P FP MP-F at 30-120 days. Normal adipocytes were obviously decreased in the control groups with increased granulation tissues, whereas normal adipocytes with capillary formations were maintained in the F/P MP-F group. Thus, adding F/P MP-F to subdivided fat grafts helps to improve graft volume retention and survival in soft-tissue reconstruction through accelerating adipocyte-survival rates and angiogenesis.

Three-Dimensional Expansion Using Plasma-Medium Gel with Fragmin/Protamine Nanoparticles and FGF-2 to Stimulate Adipose-Derived Stromal Cells and Bone Marrow-Derived Mesenchymal Stem Cells

Abstract Fragmin/protamine nanoparticles (F/P NPs) have been used as carriers for the preservation and controlled release of fibroblast growth factor (FGF)-2 and various cytokines in human plasma (HP). This study tested an HP–Dulbeccos modified Eagles medium (DMEM) gel as a three-dimensional (3D) culture for the expansion of adipose tissue-derived multilineage stromal cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs). The growth of these cells improved in 3D culture using low-concentration HP (2%)–DMEM gel with 0.1 mg/mL F/P NPs and 5 ng/mL FGF-2 without animal serum in comparison to two-dimensional (2D) culture using a low-concentration human serum (2%)–DMEM containing 5 ng/mL FGF-2 on F/P NPs-coated plates. ASCs and BMSCs, which were expanded in the low-concentration HP–DMEM gel with F/P NPs and FGF-2, maintained their multilineage potential for differentiation into adipocytes or osteoblasts similar to the 2D cultured cells. Furthermore, flow cytometric analyses showed that the phenotypic markers which were positive for CD44, CD90, and CD105 (>80%) and negative for CD34 and CD45 (<1%) were well maintained in both 2D and 3D cultures after 7 days. Thus, this 3D culture system in low-concentration HP–DMEM gel with F/P NPs and FGF-2 provided an effective and safe method for the expansion of both cell types without using animal serum.