Alginate hydrogel as a potential alternative to hyaluronic acid as submucosal injection material.

BACKGROUND: Sodium alginate is currently used in medical products, including drugs and cosmetic materials. It can also be used as a submucosal injection material due to its excellent water retention ability. Alginate with a high water retention ability is called alginate hydrogel (AH). The aim of this study was to investigate the usefulness of AH as a submucosal injection material. METHODS: To investigate the optimal viscosity of AH as a submucosal injection material, we observed the changes in submucosal height from the initial submucosal height in the stomachs of six miniature pigs for each injection material tested (0.3 % AH, 0.5 % hyaluronic acid, glycerol). All submucosal heights were compared serially over time (3, 5, 10, 20, and 30 min). Both immediate and 1-week delayed tissue reactions were investigated endoscopically in the same living pigs. Histological analyses were performed after the animals had been sacrificed. RESULTS: In a preliminary study, we determined that 0.3 % sodium alginate mixed with BaCl2 (400 µl) was the optimal viscosity of AH as an injection material. Our comparison of submucosal height changes over time showed that there was a significant decrease in submucosal height just 3 min following the injection of hyaluronic acid and glycerol, but that following the injection of AH a significant decrease in submucosal height was observed only after 10 min (p < 0.05). The histological analyses revealed that there were mild capillary dilations with congestion and mild fibrotic changes with some lymphocytic infiltration at the AH injection site. CONCLUSION: Alginate hydrogel demonstrated long-lasting maintenance of submucosal elevation, safety, and cost-effectiveness in a pig model, which makes it a potential alternative to hyaluronic acid.

Functional nanoparticles translocation into cell and adhesion force curve analysis.

The aim of this research is to investigate the cell translocation of two functional nanoparticles (barium sulfate (BaSO4NPs), europium (III) doped gadolinium oxide nanoparticles (Gd2O3@EuNPs)) into A549 cells by Bio-Atomic Force Microscopy (Bio-AFM). Successful cell translocation of these two nanoparticles are ensured from the measurement of changes in the cell surface roughness and interaction (extension), retraction forces from the vertical deflection of tip towards substrate surfaces through force-distance curve slope analysis. Measurement of typical adhesion forces (i.e., extension and retraction) between the tip-substrate (0.0963 and 1.155 nN), tip-A549 cell substrate (0.1177 and 2.468 nN), tip-Gd2O3@EuNPs/A549 substrate (0.0785 and 0.4276 nN) and tip-BaSO4NPs/A549 substrate (0.518 and 6.838 nN) confirms the successful cell translocation of functional nanoparticles into A549 cells. Further the nanoscale resolution of topographical height and 3D images evinces the surface characteristics of normal A549 cells and nanoparticles translocated A549 cells.

In vivo specific delivery of c-Met siRNA to glioblastoma using cationic solid lipid nanoparticles.

RNA interference is a powerful strategy that inhibits gene expression through specific mRNA degradation. In vivo, however, the application of small interfering RNAs (siRNAs) is severely limited by their instability and their poor delivery into target cells and tissues. This is especially true with glioblastomas (GBMs), the most frequent and malignant form of brain tumor, that has limited treatment options due to the largely impenetrable blood-brain barrier. Here, cationic solid lipid nanoparticles (SLN), reconstituted from natural components of protein-free low-density lipoprotein, was conjugated to PEGylated c-Met siRNA. The c-Met siRNA-PEG/SLN complex efficiently down-regulated c-Met expression level, as well as decreased cell proliferation in U-87MG in vitro. In orthotopic U-87MG xenograft tumor model, intravenous administration of the complex significantly inhibited c-Met expression at the tumor tissue and suppressed tumor growth without showing any systemic toxicity in mice. Use of Cy5.5 conjugated SLN revealed enhanced accumulation of the siRNA-PEG/SLN complexes specifically in the brain tumor. Our data demonstrates the feasibility of using siRNA-PEG/SLN complexes as a potential carrier of therapeutic siRNAs for the systemic treatment of GBM in the clinic.

PEGylated polyethyleneimine grafted silica nanoparticles: enhanced cellular uptake and efficient siRNA delivery.

The present paper reports the utilization of hybrid nanocomposite particles consisting of PEI25k-PEG5k copolymer grafted silica nanoparticles (SiO(2)NPs) for enhanced cellular uptake and siRNA delivery. High-resolution transmission electron microscopy and dynamic light scattering measurements ensured the average particle size of the final hybrid component as 45 nm (core SiO(2), 28-30 nm and shell PEI25k-PEG5k, 12-15 nm). Surface morphology from atomic force microscopy analysis showed the significant relationship between the particle size and shape. (29)Si and (13)C cross-polarization-magic angle spinning solid state nuclear magnetic resonance (NMR), (1)H-NMR, and Fourier transform infrared spectroscopy were used to obtain the relevant structural information (such as Q3, silanol; Q4, siloxane functional groups of SiO(2)NPs; resonance shifts and bending vibrations of PEI25k, -CH(2)-CH(2)-NH-; and PEG5k, -CH(2)-CH(2)-O-) from copolymer nanoparticle. Stable complexation of siRNA and nanocomposite particle (wt.%:wt.%) was achieved from 1:5 to 1:15 ratio. Nanocomposite particle (N/P) ratio and siRNA concentration determine the stability and knockdown efficiency of the PEI25k-PEG5k-graft-SiO(2)NPs-siRNA complexes. It was shown that highly positively charged (zeta potential, +66 mV) PEI25k-PEG5k-graft-SiO(2)NPs result in strong affinity with negatively charged siRNA. Confocal microscopy showed intensified cellular uptake of siRNA into cytoplasm of A549 cancer cell utilized for in vitro study. In conclusion, the coherence, graft density of copolymer-SiO(2)NPs, and siRNA concentration were found to strongly influence the stability, and hence determine the knockdown efficiency, of PEI25k-PEG5k-graft-SiO(2)NPs-siRNA complexes.

Structural and biological evaluation of a multifunctional SWCNT-AgNPs-DNA/PVA bio-nanofilm.

A bio-nanofilm consisting of a tetrad nanomaterial (nanotubes, nanoparticles, DNA, polymer) was fabricated utilizing in situ reduction and noncovalent interactions and it displayed effective antibacterial activity and biocompatibility. This bio-nanofilm was composed of homogenous silver nanoparticles (AgNPs) coated on single-walled carbon nanotubes (SWCNTs), which were later hybridized with DNA and stabilized in poly(vinyl alcohol) (PVA) in the presence of a surfactant with the aid of ultrasonication. Electron microscopy and bio-AFM (atomic force microscopy) images were used to assess the morphology of the nanocomposite (NC) structure. Functionalization and fabrication were examined using FT-Raman spectroscopy by analyzing the functional changes in the bio-nanofilm before and after fabrication. UV-visible spectroscopy and X-ray powder diffraction (XRD) confirmed that AgNPs were present in the final NC on the basis of its surface plasmon resonance (370 nm) and crystal planes. Thermal gravimetric analysis was used to measure the percentage weight loss of SWCNT (17.5%) and final SWCNT-AgNPs-DNA/PVA (47.7%). The antimicrobial efficiency of the bio-nanofilm was evaluated against major pathogenic organisms. Bactericidal ratios, zone of inhibition, and minimum inhibitory concentration were examined against gram positive and gram negative bacteria. A preliminary cytotoxicity analysis was conducted using A549 lung cancer cells and IMR-90 fibroblast cells. Confocal laser microscopy, bio-AFM, and field emission scanning electron microscopy (FE-SEM) images demonstrated that the NCs were successfully taken up by the cells. These combined results indicate that this bio-nanofilm was biocompatible and displayed antimicrobial activity. Thus, this novel bio-nanofilm holds great promise for use as a multifunctional tool in burn therapy, tissue engineering, and other biomedical applications.

Possibility of skin epithelial cell transdifferentiation in tracheal reconstruction.

In tissue engineering, injured tissue is normally reconstructed with cells obtained from that tissue itself. However, it is difficult to obtain cells for reconstruction of the trachea because of its shape and limited accessibility. Therefore, other cell sources having similar form and function or stem cells are used for tracheal reconstruction. In a previous study, we used autologous skin epithelial cells and successfully reconstructed canine tracheas. We found that the tracheal epithelial layer was completely covered with ciliated cells, which is a remarkable finding because skin and tracheal epithelial cells originate from different germinal layers and have very different forms. In this study, to elucidate the origin of the ciliated cells, we identified the stem cell contents of skin epithelial cells on primary culture, marked the skin epithelial cells with PKH26 dye, and transplanted them onto canine tracheas. After 5 months, we identified PKH26 fluorescence on the tracheal epithelial layers, especially over the tracheal cartilages. Consequently, we demonstrated that transplanted autologous skin epithelial stem cells can remain viable on the trachea for a few months and can transdifferentiate into tracheal epithelial cells and chondrocytes.

Antiobesity effects and improvement of insulin sensitivity by 1-deoxynojirimycin in animal models.

The alpha-glucosidase inhibitor 1-deoxynojirimycin (DNJ) is one of the simplest naturally occurring carbohydrate mimics, with promising biological activity in vivo. Although there is considerable interest in the pharmacological effects of DNJ, the antidiabetic effects of DNJ in type 2 diabetes mellitus have received little attention. In this work, DNJ was isolated from the silkworm (Bombyx mori), and its antidiabetic effects were evaluated in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an established animal model of human type 2 diabetes mellitus, and in control Long-Evans Tokushima Otsuka (LETO) rats. DNJ treatment showed significant antidiabetic effects in OLETF rats, with significant improvements in fasting blood glucose levels and glucose tolerance and, especially, increased insulin sensitivity. Furthermore, there was significant loss of body weight in both groups. DNJ also showed significant antihyperglycemic effects in streptozotocin- and high-fat-diet-induced hyperglycemic rats. Its efficacy and dose profiles were better than those of acarbose, a typical alpha-glucosidase inhibitor in clinical use. Furthermore, a substantial fraction of DNJ was absorbed into the bloodstream within a few minutes of oral administration. DNJ was also detected in the urine. These findings suggest that its postprandial hypoglycemic effect in the gastrointestinal tract is a possible but insufficient mechanism of action underlying the antidiabetic effects of DNJ. Its antiobesity effect and improvement of insulin sensitivity are other possible antidiabetic effects of DNJ.

Nanoparticulate carrier containing water-insoluble iodinated oil as a multifunctional contrast agent for computed tomography imaging.

Contrast-enhanced computed tomography (CT) imaging is a valuable and routine strategy for the clinical diagnosis of various diseases. However, all current CT contrast agents are liquids, so they flow through the blood vessels and disappear very quickly by extravasation. If it were possible to make a blood-compatible particulate contrast agent, we could highlight a particular tissue by either passive or active targeting. In this work, Pluronic F127 and a naturally iodinated compound, Lipiodol, were used to form radiopaque nanoreservoir structures. The resultant nanoparticles have a stable structure at high concentrations, sufficient X-ray absorption, a safety profile similar to or better than that of Iopromide, and a longer circulation time than commercial iodinated preparations. The utility of the resultant radiopaque nanoparticles as a contrast agent was tested using micro-SPECT/CT imaging in vivo. Together with the very good solubility of hydrophobic drugs (e.g., Taxol) in Lipiodol, these results suggest the possibility that these particulate structures and their bioconjugates could become functional CT contrast agents that could deliver therapeutic agents to a particular tissue.

In situ synthesis of silver nanoparticles on the surface of PDMS with high antibacterial activity and biosafety toward an implantable medical device.

We developed a straightforward method to fabricate antibacterial silicon films via the in situ synthesis of silver nanoparticles (AgNPs) on a polydimethylsiloxane (PDMS) film. To grow AgNPs attached on the film, AgNP seeds were synthesized through the reduction of silver ions electrostatically bound to hydroxyl groups formed on the surface of the film after treatment with air plasma. In the growth reaction, silver ions were reduced on the seeds of AgNPs by sodium citrate in a solution of AgNO3, which allowed for the formation of AgNPs with sizes of up to ~ 500 nm, which The formed AgNPs on the films were characterized using UV-vis spectrophotometer, scattering electron microscope and induced coupled mass spectrometer. The amount of AgNPs was estimated to be less than 0.05% of the total film weight. Even though it was coated with a small amount of AgNPs, the PDMS film exhibited reduction of E. coli and S. aureus with values of log10 4.8 and log10 5.7, respectively. The biosafety of the AgNP-attached PDMS film was examined by contact of cells with the film or film eluent. Counting of viable cells revealed no significant cytotoxicity of the in situ-fabricated AgNPs on the PDMS film.

Silica nanoparticle-based dual imaging colloidal hybrids: cancer cell imaging and biodistribution.

In this study, fluorescent dye-conjugated magnetic resonance (MR) imaging agents were investigated in T mode. Gadolinium-conjugated silica nanoparticles were successfully synthesized for both MR imaging and fluorescence diagnostics. Polyamine and polycarboxyl functional groups were modified chemically on the surface of the silica nanoparticles for efficient conjugation of gadolinium ions. The derived gadolinium-conjugated silica nanoparticles were investigated by zeta potential analysis, transmission electron microscopy, inductively coupled plasma mass spectrometry, and energy dispersive x-ray spectroscopy. MR equipment was used to investigate their use as contrast-enhancing agents in T1 mode under a 9.4 T magnetic field. In addition, we tracked the distribution of the gadolinium-conjugated nanoparticles in both lung cancer cells and organs in mice.

Factors affecting tissue culture and transplantation using omentum.

In tissue engineering, a stable tissue layer and blood vessels are required for complete tissue formation, to provide structural strength and thickness and to supply oxygen and various nutrients. However, this has not been achieved using in vitro tissue-engineered culture techniques, thus many tissue engineering studies of trachea, bladder, and intestine reconstruction have used omentum. However, many factors critical to cell culture and transplantation using omentum have not yet been studied. For these reasons, we conducted a study of artificial trachea reconstruction in dogs using a poly(lactic-co-glycolic acid) (PLGA) porous scaffold, polypropylene prosthesis, and PKH26-labeled cells. We analyzed factors affecting tissue-engineered reconstruction using omentum, such as cell distribution and formation of cell layer and stability of transplant shape on omentum. As a result, we classified failure factors for tissue-engineered application of omentum and suggested three considerations for effective use of omentum in tissue engineering. Our observations may aid in the design and execution of future studies of omentum usage in tissue engineering.

Rapid detection of the epidermal growth factor receptor mutation in non-small-cell lung cancer for analysis of acquired resistance using molecular beacons.

A secondary mutation (T790M) in epidermal growth factor receptor (EGFR) is a hallmark of acquired resistance to EGFR inhibitors used to treat non-small-cell lung cancer (NSCLC). Therefore, identifying the T790M mutation is crucial to guide treatment decisions. Given that DNA sequencing methods are time-consuming and insensitive, we developed and investigated the feasibility of using molecular beacons for the detection of the T790M mutation in EGFR. A molecular beacon complementary to the region of the secondary EGFR mutation (T790M) was designed and used in NSCLC samples bearing drug-sensitive and -resistant EGFR mutations. For a rapid and simple assay, we attempted to use the molecular beacon with real-time PCR and in situ fluorescence imaging. The ability of the designed molecular beacon to specifically detect the T790M mutation of EGFR was tested for samples from two patients with drug resistance and compared with conventional DNA sequencing methods. The molecular beacon successfully detected the T790M mutation in patient samples with drug resistance. The sensitivity of the molecular beacon, which detected as little as 2% of genomic DNA from the drug-resistant cells (H1975), was much higher than direct sequencing. Furthermore, in situ fluorescence imaging with the molecular beacon gave rise to a distinguishable signal for the T790M mutation in drug-resistant cells. The molecular beacon-based approach enabled rapid and sensitive detection of the EGFR mutation (T790M) in NSCLC with in situ fluorescence imaging, which can be directed to determine various treatment options in patients with cancer.

Schedule-dependent synergistic effect of rituximab on methotrexate chemotherapy against lymphoma of the central nervous system.

We hypothesized that methotrexate (MTX) normalizes the increased permeability of the blood-tumor barrier and thus reduces the accessibility of rituximab (RTX) to central nervous system (CNS) lymphoma. Here, we evaluated the combinational treatment capability of RTX and MTX using an alternative treatment schedule against CNS lymphoma. We developed a CNS lymphoma animal model that closely mimics the morphological and molecular characteristics of human CNS lymphoma by injecting Raji human Burkitt lymphoma cells into the brains of immune-compromised mice and tested a novel combinational treatment schedule by which penetration of RTX was not influenced by MTX administration. RTX was conjugated with Alexa Fluor 680, and its distribution in the brain was analyzed by in vivo imaging. When MTX treatment was followed by a 3-day post RTX administration, RTX was scarcely distributed in the brain, and there were only modest statistically insignificant therapeutic effects compared with the control mice which received sham injections. In contrast, RTX administration followed by a 3-day post MTX treatment showed significantly increased distribution of RTX and significantly reduced tumor volume in the brain. Collectively, our data demonstrate that RTX can be successfully combined with MTX using an alternative treatment schedule that allows increased distribution of RTX in CNS lymphoma.

Modest anti-cancer activity of a bile acid acylated heparin derivative in a PC14PE6 induced orthotopic lung cancer model.

PURPOSE: A novel chemically modified heparin derivative, heparin-deoxycholic acid nano-particles, has lower anticoagulant activity, and was recently reported to have significant anti-tumor effects on squamous head and neck cancer cells. Therefore, the aim of this study was to evaluate the anti-tumor effects of heparin-deoxycholic acid nano-particles in a human lung adenocarcinoma cell line. MATERIALS AND METHODS: An orthotopic lung cancer model in 16 mice was developed using intra-thoracic injections of 0.5x10(6) PC14PE6 cells. Ten days after inoculation, the mice were divided into two groups. PBS and Heparin-DOCA particles were injected once a day every 3 days in the tail vein, for a total of 5 injections. The body weight and survival of each mouse were monitored and the tumor size in the lung was measured by SPECT-CT before and after heparin-DOCA nano-particle treatment. RESULTS: IThe HD particles had no significant cytotoxicity when the PC9 cells were treated in vitro. There was no statistical difference in tumor size, body weight and survival between the HD treated and control groups in vivo. Furthermore, there was no difference in the amount of CD31 between tumor tissues in the two study groups. CONCLUSION: HD synthesized with unfractionated heparin had no apparent inhibitory effects on tumor growth in a PC14PE6 cell induced orthotopic lung cancer mouse model. The HD particles did not significantly inhibit tumor-induced angiogenesis at the tumor sites.

Efficient intracellular siRNA delivery strategy through rapid and simple two steps mixing involving noncovalent post-PEGylation.

Two different and well-defined methacrylate-based (co)polymers were employed as a polymeric siRNA delivery system. siRNA, poly(2-(dimethylamino) ethyl methacrylate) homopolymers (PDMAEMA) and poly(2-(dimethylamino) ethyl methacrylate)-b-poly (ethyleneglycol) alpha-methoxy, omega-methacrylate (PDMAEMA-b-PMAPEG) palm-tree-like copolymer ternary complexes were prepared using a rapid and simple two-step mixing protocol involving noncovalent post-PEGylation, and physicochemical properties including hydrodynamic diameter, zeta-potential and siRNA condensation efficiency were characterized. Transfection efficiency, intracellular uptake, and cytotoxicity of ternary complexes were also evaluated. Ternary complexes provide efficient condensation and compaction of siRNA within the cationic core of complexes. Noncovalent post-PEGylation provides the ternary complexes with enzymatic and serum stability without harming complex formation and condensation of siRNA. Thereby, under an optimal N/P ratio, ternary complexes exhibited brilliant gene silencing efficiency with low cytotoxicity in media containing 10% serum. Confocal microscopy clearly showed efficient and even intracellular uptake of complexes by cells via endocytosis. This study highlights the excellent properties of noncovalent post-PEGylated ternary complexes produced by rapid and simple mixing. Accordingly, these findings suggest that the formation of ternary complexes could be utilized as a safe and effective polymeric siRNA delivery strategy.