Local Gene Delivery System by Bubble Liposomes and Ultrasound Exposure into Joint Synovium

Recently, we have developed novel polyethylene glycol modified liposomes (bubble liposomes; BL) entrapping an ultrasound (US) imaging gas, which can work as a gene delivery tool with US exposure. In this study, we investigated the usefulness of US-mediated gene transfer systems with BL into synoviocytes in vitro and joint synovium in vivo. Highly efficient gene transfer could be achieved in the cultured primary synoviocytes transfected with the combination of BL and US exposure, compared to treatment with plasmid DNA (pDNA) alone, pDNA plus BL, or pDNA plus US. When BL was injected into the knee joints of mice, and US exposure was applied transcutaneously to the injection site, highly efficient gene expression could be observed in the knee joint transfected with the combination of BL and US exposure, compared to treatment with pDNA alone, pDNA plus BL, or pDNA plus US. The localized and prolonged gene expression was also shown by an in vivo luciferase imaging system. Thus, this local gene delivery system into joint synovium using the combination of BL and US exposure may be an effective means for gene therapy in joint disorders

Cellulose Chemistry Meets Click Chemistry: Syntheses and Properties of Cellulose-Based Glycoclusters with High Structural Homogeneity

b-1,4-Glucans having oligosaccharide appendages (O-/N-linked b-maltoside and O-/N-linked b-lactoside) at 6C positions of all repeating units can be readily prepared from cellulose through a two step strategy composed of: (1) regio-selective and quantitative bromination/azidation to afford 6-azido-6-deoxycellulose; and (2) the subsequent Cu+-catalyzed coupling with oligosaccharides having terminal alkyne. The resultant cellulose derivatives showed improved water solubility in comparison to native cellulose; they, however, bound to carbohydrate-binding proteins in a rather non-specific manner. Molecular dynamics calculations revealed that these properties are attributable to rigid sheet-like structures of the cellulose derivatives and the subsequent exposure of their hydrophobic moieties to solvents

Structure-Activity Relationship of Mono-Ion Complexes for Plasmid DNA Delivery by Muscular Injection

The structure-activity relationship of mono-ion complexes (MICs) for plasmid DNA (pDNA) delivery by muscular injection is demonstrated. MICs were formed between pDNA and monocationic poly(ethylene glycol) (PEG) macromolecules. As monocationic PEGs, the ω-amide-pentylimidazolium (APe-Im) end-modified PEGs with a stable amide (Am) and hydrolytic ester (Es) bond, that is, APe-Im-Am-PEG and APe-Im-Es-PEG, respectively, are synthesized. The difference between the APe-Im-Am-PEG and APe-Im-Es-PEG was only a spacer structure between a terminal cation and a PEG chain. The resulting pDNA MICs with APe-Im-Am-PEG at a charge ratio (+/−) of 32 or 64 were more stable than those with APe-Im-Es-PEG in the presence of serum proteins. The highest gene expression by muscular injection was achieved using the APe-Im-Am-PEG/pDNA MIC at a charge ratio (+/−) of 32 with a smaller particle diameter of approximately 50 nm, as compared to that charge ratio of 64. Consequently, the pDNA MIC with the monocationic PEG with a stable amide spacer, as compared to a hydrolytic ester spacer, is considered to be suitable for the highest gene expression by muscular injection

Development of porous solid reactant for thermal-energy storage and temperature upgrade using carbonation/decarbonation reaction

Cyclic reaction performances of solid reactants for a CaO-CO2 chemical heat-pump designed for upgrading and storing high-temperature thermal energy were studied. Solid reactants composed of CaO as the reactant and CaTiO3 as the inert framework were prepared using the conventional powder method or the metal alkoxide method. Upon experiments of cyclic operation between CaO carbonation and CaCO3 decarbonation at 1023K, the reaction reversibility of the solid reactants with the inert CaTiO3 framework was steady, whereas that of the solid reactant without the inert framework decreased with sintering of the solid particles during cyclic operation. Reaction rates for the first carbonation and the decarbonation of solid reactant prepared using the alkoxide method were about 1.8 and 2.4 times faster, respectively, than for those prepared by the powder method due to the smaller average diameter of reactant particles derived from the alkoxide method.

Byproduct-Free Intact Modification of Insulin by Cholesterol End-Modified Poly(ethylene glycol) for in Vivo Protein Delivery

Insulin is a key peptide hormone used for the treatment of both type I and type II diabetes. To maximize the effect of the treatment of these diseases, the addition of poly­(ethylene glycol) (PEGylation) methods for the insulin are widely developed. Here, to make these PEGylation methods the simplest, we report the byproduct-free intact modification of insulin by cholesterol end-modified poly­(ethylene glycol) with urethane, propyl, and methoxy groups (that is, Chol–U–Pr–mPEG). The noncovalent PEGylation by the Chol–U–Pr–mPEG has been achieved by the simple mixing of insulin with the Chol–U–Pr–mPEG in aqueous solution, followed by freeze-drying. The formation of the Chol–U–Pr–mPEG/insulin complex has proceeded without byproducts, such as <i>N</i>-hydroxysuccinimide, formed by the conventional covalent PEGylation using an active ester group. The byproduct-free PEGylation has preserved insulin conformation as well as primary structure. The intact PEGylation has protected insulin from hydrolysis by protease. The resulting insulin modified by the Chol–U–Pr–mPEG has sustainably suppressed the level of blood glucose, as compared to naked insulin, in mice. Consequently, the Chol–U–Pr–mPEG/insulin complex formation offers the byproduct-free intact PEGylation of insulin for in vivo protein delivery

Role of Interleukin-6 in the Antigen-Specific Mucosal Immunoglobulin A Responses Induced by CpG Oligodeoxynucleotide-Loaded Cationic Liposomes

An advantage of mucosal vaccines over conventional parenteral vaccines is that they can induce protective immune responses not only at mucosal surfaces but also in systemic compartments. Despite this advantage, few live attenuated or inactivated mucosal vaccines have been developed and applied clinically. We recently showed that the intranasal immunization of ovalbumin (OVA) with class B synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motif (CpG ODN)-loaded cationic liposomes synergistically exerted both antigen-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) responses in mice. However, the mechanism underlying the mucosal adjuvant activity of CpG ODN-loaded liposomes remains unknown. In the present study, we showed that the intranasal administration of CpG ODN-loaded cationic liposomes elicited interleukin (IL)-6 release in nasal tissues. Additionally, pre-treatment with an anti-IL-6 receptor (IL-6R) antibody attenuated antigen-specific nasal IgA production but not serum IgG responses. Furthermore, the intranasal administration of OVA and CpG ODN-loaded cationic liposomes increased the number of IgA+/CD138+ plasma cells and IgA+/B220+ B cells in the nasal passages. This increase was markedly suppressed by pre-treatment with anti-IL-6R blocking antibody. In conclusion, IL-6 released by CpG ODN-loaded cationic liposomes at the site of administration may play a role in the induction of antigen-specific IgA responses by promoting differentiation into IgA+ plasma cells for IgA secretion from B cells

Surface Phenotype Changes and Increased Response to Oxidative Stress in CD4+CD25high T Cells

Conversion of CD4+CD25+FOXP3+ T regulatory cells (Tregs) from the immature (CD45RA+) to mature (CD45RO+) phenotype has been shown during development and allergic reactions. The relative frequencies of these Treg phenotypes and their responses to oxidative stress during development and allergic inflammation were analysed in samples from paediatric and adult subjects. The FOXP3lowCD45RA+ population was dominant in early childhood, while the percentage of FOXP3highCD45RO+ cells began increasing in the first year of life. These phenotypic changes were observed in subjects with and without asthma. Further, there was a significant increase in phosphorylated ERK1/2 (pERK1/2) protein in hydrogen peroxide (H2O2)-treated CD4+CD25high cells in adults with asthma compared with those without asthma. Increased pERK1/2 levels corresponded with increased Ca2+ response to T cell receptor stimulation. mRNA expression of peroxiredoxins declined in Tregs from adults with asthma. Finally, CD4+CD25high cells from paediatric subjects were more sensitive to oxidative stress than those from adults in vitro. The differential Treg sensitivity to oxidative stress observed in children and adults was likely dependent on phenotypic CD45 isoform switching. Increased sensitivity of Treg cells from adults with asthma to H2O2 resulted from a reduction of peroxiredoxin-2, -3, -4 and increased pERK1/2 via impaired Ca2+ response in these cells