Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.     

Microbead-based electrochemical immunoassay with interdigitated array electrodes

The objective of this study was to develop a sensitive and miniaturized immunoassay by coupling a microbead-based immunoassay with an interdigitated array (IDA) electrode. An IDA electrode amplifies the signal by recycling an electrochemically redox-reversible molecule. The microfabricated platinum electrodes had 25 pairs of electrodes with 1.6-microm gaps and 2.4-microm widths. An enzyme-labeled sandwich immunoassay on paramagnetic microbeads with mouse IgG as the analyte and beta-galactosidase as the enzyme label was used as the model system. beta-Galactosidase converted p-aminophenyl beta-D-galactopyranoside to p-aminophenol (PAP). This enzyme reaction was measured continuously by positioning the microbeads near the electrode surface with a magnet. Electrochemical recycling occurred with PAP oxidation to p-quinone imine (PQI) at +290 mV followed by PQI reduction to PAP at -300 mV vs Ag/AgCl. Dual-electrode detection amplified the signal fourfold compared to single-electrode detection, and the recycling efficiency reached 87%. A calibration curve of PAP concentration vs anodic current was linear between 10(-4) and 10(-6)M. A signal from 1000 beads in a 20-microL drop was detectable and the immunoassay was complete within 10 min with a detection limit of 3.5x10(-15)mol mouse IgG.     

Activated sludge encapsulation in gellan gum microbeads for gasoline biodegradation

A two-phase dispersion technique, termed emulsification–internal gelation, is proposed for encapsulation of activated sludge in gellan gum microbeads. The influence of emulsion parameters on size distribution of microbeads was investigated. Mean diameter of microbeads varied within a range of 34–265 µm as a descending function of emulsion stirring rate (1,000–5,000 rpm), emulsification time (10–40 min), and emulsifier concentration (0–0.1% w/w), and as an ascending function of disperse phase volume fraction (0.08–0.25). Encapsulated sludge expressed a high biodegradation activity compared with non-encapsulated sludge cultures even at 4.4 times lower level of overall biomass loading. Over 90% of gasoline at an initial concentration of 35 and 70 mg l^–1 was removed by both encapsulated and non-encapsulated sludge cultures in sealed serum bottles within 7 days. Encapsulation of activated sludge in gellan gum microbeads enhanced the biological activity of microbial populations in the removal of gasoline hydrocarbons. The results of this study demonstrated the feasibility of the production of size-controlled gellan gum-encapsulated sludge microbeads and their use in the biodegradation of gasoline.     

Bone morphogenetic protein-4 and Noggin signaling regulates pigment cell distribution in the axolotl trunk

Abstract Wild-type (dark) and white mutant axolotl ( Ambystoma mexicanum ) embryos were used to investigate the role of the secreted growth factor bone morphogenetic protein-4 (BMP-4) and its antagonist, Noggin, in dorso-lateral trunk neural crest (NC) migration. Implantation of a BMP-4-coated microbead caused a melanophore-free zone around the bead, reduction of the dorsal fin above the bead, and disappearance of myotome tissue. We established a novel method that allows controlled induction of protein synthesis and release. Xenopus animal cap (XAC) cells injected with heat shock-inducible constructs for BMP-4 and Noggin were implanted into axolotl embryos and protein expression was induced at defined time points. With this approach, we could demonstrate for the first time that Noggin can stimulate melanophore migration in the white mutant. We further showed that implantation of BMP-4 expressing XAC cells alters pigment cell distribution without affecting muscle and dorsal fin development.     

Characterisation of tyrosinase immobilised onto spacer-arm attached glycidyl methacrylate-based reactive microbeads

Immobilisation of tyrosinase onto modified poly(methyl methacrylate–glycidyl methacrylate–divinyl benzene), poly(MMA–GMA–DVB), microbeads was studied. The epoxy group containing poly(MMA–MMA–DVB) microbeads were prepared by suspension polymerisation. The epoxy groups of the poly(MMA–GMA–DVB) microbeads was converted into amino groups with either ammonia or 1,6-diaminohexane (i.e., spacer-arm). Tyrosinase was then covalently immobilised on aminated and the spacer-arm-attached poly(MMA–GMA–DVB) microbeads using glutaric dialdehyde as a coupling agent. Incorporation of the spacer-arm resulted an increase in the apparent activity of the immobilised tyrosinase with respect to the enzyme immobilised on the aminated microbeads. The activity yield of the immobilised tyrosinase on the spacer-arm-attached poly(MMA–GMA–DVB) microbeads was 68%, and this was 51% for the enzyme, which was immobilised on the aminated microbeads. Both immobilised tyrosinase preparation has resistance to temperature inactivation as compared to that of the free form. The temperature profiles were broader for both immobilised preparations than that of the free enzyme. Kinetic parameters were determined for immobilised tyrosinase preparations as well as for the free enzyme. The values of the Michaels constants (K_m) for all the immobilised tyrosinase preparations were significantly larger, indicating decreased affinity by the enzyme for its substrate, whereas V_max values were smaller for the both immobilised tyrosinase preparations. In a 40 h continuous operation with spacer-arm-attached poly(MMA–GMA–DVB) microbeads at 30 °C, only 3% of immobilised tyrosinase activity was lost. The operational inactivation rate constant (k_opi) of the immobilised tyrosinase was 1.25×10⁻⁵ min⁻¹.     

Phagocytosis of Protein Coated Colloidal-Gold-Agarose-Gelatin Microbeads by Cultured Uterine Glandular Epithelial and Stromal Cells

A procedure for fixing and immunostaining whole cells from primary cultures of ovine and bovine uterine gland fragments was used to identify keratin in intermediate filaments of epithelial cells to distinguish them from stromal cells. Colloidal gold encapsulated aga-rose-gelatin microbeads were coated with different proteins and used to investigate uptake by epithelial and stromal cells in culture. Micro-beads were taken up by stromal cells and by epithelial cells on the outskirts of colonies. These cells formed ridges where they contacted and grew above stromal cells. Electron microscopy demonstrated that the microbeads had been internalized and appeared to be nontoxic. Individual cells could harbor more than 90 microbeads within their cytoplasm for at least seven to ten days with no apparent harm. Some cells with microbeads were seen to divide.     

Comparative proteomic analysis of primary mouse liver c-Kit-(CD45/TER119)- stem/progenitor cells

Liver stem/progenitor cells play a key role in liver development and maybe also in liver cancer development. In our previous study a population of c-Kit-(CD45/TER119)- liver stem/progenitor cells in mouse fetal liver, was successfully sorted with large amount (10(6)-10(7)) by using immuno-magnetic microbeads. In this study, the sorted liver stem/progenitor cells were used for proteomic study. Proteins of the sorted liver stem/progenitor cells and unsorted fetal liver cells were investigated using two-dimensional electrophoresis. A two-dimensional proteome map of liver stem/progenitor cells was obtained for the first time. Proteins that exhibited significantly upregulation in liver stem/progenitor cells were identified by peptide mass fingerprinting and peptide sequencing. Nineteen protein spots corresponding to 12 different proteins were identified as showing significant upregulation in liver stem/progenitor cells and seem to play important roles in such cells in cell metabolism, cell cycle regulation, and stress. An interesting finding is that most of the upregulated proteins were overexpressed in various cancers (11 of 12, including 6 in human hepatocellular carcinoma (HCC)) and involved in cancer development as reported in previous studies. Some of the identified proteins were validated by real-time PCR, Western blotting, and immunostaining. Taken together, the data presented provide a significant new protein-level insight into the biology of liver stem/progenitor cells, a key population of cells that might be also involved in liver cancer development.     

New sorbent for bilirubin removal from human plasma: Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads

Cibacron Blue F3GA-immobilized poly(EGDMA–HEMA) microbeads were investigated as a specific sorbent for bilirubin removal from human plasma. The poly(EGDMA–HEMA) microbeads were prepared by a modified suspension copolymerization technique. Cibacron Blue F3GA was covalently coupled to the poly(EGDMA–HEMA) microbeads via the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of the HEMA molecule, under alkaline conditions. Bilirubin adsorption was investigated from hyperbilirubinemic human plasma on the poly(EGDMA–HEMA) microbeads containing different amounts of immobilized Cibacron Blue F3GA, (between 5.0–16.5 μmol/g). The non-specific bilirubin adsorption on the unmodified poly(EGDMA–HEMA) microbeads were 0.32 mg/g from human plasma. Higher bilirubin adsorption values, up to 14.8 mg/g, were obtained with the Cibacron Blue F3GA-immobilized microbeads. Bilirubin molecules interacted with these sorbents directly. Contribution of albumin adsorption on the bilirubin adsorption was pronounced. Bilirubin adsorption increased with increasing temperature.