Multi-responsive hydrogels based on N-isopropylacrylamide and sodium alginate

Hydrogels consisting of sodium alginate and N-isopropylacrylamide covalently crosslinked with N,N′-methylenebisacrylamide were prepared. The mixed-interpenetrated networks obtained were characterized using elemental analysis, Fourier transform infrared and Raman spectroscopy, swelling measurements and environmental scanning electron microscopy. The thermo- and pH-responsive properties of these hydrogels were evidenced by their swelling behaviour, which depended also on the amount of crosslinking agent and hydrogel composition.

pH-tunable hydrogelators for water purification: structural optimisation and evaluation

A focused library of potential hydrogelators each containing two substituted aromatic residues separated by a urea or thiourea linkage have been synthesised and characterized. Six of these novel compounds are highly efficient hydrogelators, forming gels in aqueous solution at low concentrations (0.03–0.60 wt %). Gels were formed through a pH switching methodology, by acidification of a basic solution (pH 14 to ≈4) either by addition of HCl or via the slow hydrolysis of glucono-δ-lactone. Frequently, gelation was accompanied by a dramatic switch in the absorption spectra of the gelators, resulting in a significant change in colour, typically from a vibrant orange to pale yellow. Each of the gels was capable of sequestering significant quantities of the aromatic cationic dye, methylene blue, from aqueous solution (up to 1.02 g of dye per gram of dry gelator). Cryo-transmission electron microscopy of two of the gels revealed an extensive network of high aspect ratio fibers. The structure of the fibers altered dramatically upon addition of 20 wt % of the dye, resulting in aggregation and significant shortening of the fibrils. This study demonstrates the feasibility for these novel gels finding application as inexpensive and effective water purification platforms.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.

Chitosan coated alginate beads for the survival of microencapsulated Lactobacillus plantarum in pomegranate juice

This work studied the effect of multi-layer coating of alginate beads on the survival of encapsulated Lactobacillus plantarum in simulated gastric solution and during storage in pomegranate juice at 4 °C. Uncoated, single and double chitosan coated beads were examined. The survival of the cells in simulated gastric solution (pH 1.5) was improved in the case of the chitosan coated beads by 0.5–2 logs compared to the uncoated beads. The cell concentration in pomegranate juice after six weeks of storage was higher than 5.5 log CFU/mL for single and double coated beads, whereas for free cells and uncoated beads the cells died after 4 weeks of storage. In simulated gastric solution, the size of the beads decreased and their hardness increased with time; however, the opposite trend was observed for pomegranate juice, indicating that there is no correlation between cell survival and the hardness of the beads.

Development and application of a method for the purification of free shigatoxigenic bacteriophage from environmental samples

Shiga toxin producing Escherichia coli (STEC) strains are foodborne pathogens whose ability to produce Shiga toxin (Stx) is due to the integration of Stx-encoding lambdoid bacteriophage (Stx phage). Circulating, infective Stx phages are very difficult to isolate, purify and propagate such that there is no information on their genetic composition and properties. Here we describe a novel approach that exploits the phage's ability to infect their host and form a lysogen, thus enabling purification of Stx phages by a series of sequential lysogen isolation and induction steps. A total of 15 Stx phages were rigorously purified from water samples in this way, classified by TEM and genotyped using a PCR-based multi-loci characterisation system. Each phage possessed only one variant of each target gene type, thus confirming its purity, with 9 of the 15 phages possessing a short tail-spike gene and identified by TEM as Podoviridae. The remaining 6 phages possessed long tails, four of which appeared to be contractile in nature (Myoviridae) and two of which were morphologically very similar to bacteriophage lambda (Siphoviridae).

Layer-by-layer coating of alginate matrices with chitosan–alginate for the improved survival and targeted delivery of probiotic bacteria after oral administration

The oral administration of probiotic bacteria has shown potential in clinical trials for the alleviation of specific disorders of the gastrointestinal tract. However, cells must be alive in order to exert these benefits. The low pH of the stomach can greatly reduce the number of viable microorganisms that reach the intestine, thereby reducing the efficacy of the administration. Herein, a model probiotic, Bifidobacterium breve, has been encapsulated into an alginate matrix before coating in multilayers of alternating alginate and chitosan. The intention of this formulation was to improve the survival of B. breve during exposure to low pH and to target the delivery of the cells to the intestine. The material properties were first characterized before in vitro testing. Biacore™ experiments allowed for the polymer interactions to be confirmed; additionally, the stability of these multilayers to buffers simulating the pH of the gastrointestinal tract was demonstrated. Texture analysis was used to monitor changes in the gel strength during preparation, showing a weakening of the matrices during coating as a result of calcium ion sequestration. The build-up of multilayers was confirmed by confocal laser-scanning microscopy, which also showed the increase in the thickness of coat over time. During exposure to in vitro gastric conditions, an increase in viability from <3 log(CFU) per mL, seen in free cells, up to a maximum of 8.84 ± 0.17 log(CFU) per mL was noted in a 3-layer coated matrix. Multilayer-coated alginate matrices also showed a targeting of delivery to the intestine, with a gradual release of their loads over 240 min.

A novel alternative to cryopreservation for the short-term storage of stem cells for use in cell therapy using alginate encapsulation

Efficient transport of stem/progenitor cells without affecting their survival and function is a key factor in any practical cell-based therapy. However, the current approach using liquid nitrogen for the transfer of stem cells requires a short delivery time window is technically challenging and financially expensive. The present study aims to use semipermeable alginate hydrogels (crosslinked by strontium) to encapsulate, store, and release stem cells, to replace the conventional cryopreservation method for the transport of therapeutic cells within world-wide distribution time frame. Human mesenchymal stem cell (hMSC) and mouse embryonic stem cells (mESCs) were successfully stored inside alginate hydrogels for 5 days under ambient conditions in an air-tight environment (sealed cryovial). Cell viability, of the cells extracted from alginate gel, gave 74% (mESC) and 80% (hMSC) survival rates, which compared favorably to cryopreservation. More importantly, the subsequent proliferation rate and detection of common stem cell markers (both in mRNA and protein level) from hMSCs and mESCs retrieved from alginate hydrogels were also comparable to (if not better than) results gained following cryopreservation. In conclusion, this new and simple application of alginate hydrogel encapsulation may offer a cheap and robust alternative to cryopreservation for the transport and storage of stem cells for both clinical and research purposes.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Oxidized alginate hydrogels as niche environments for corneal epithelial cells

Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation

Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (Palginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins.

Microencapsulation of a synbiotic into PLGA/alginate multiparticulate gels

Probiotic bacteria have gained popularity as a defence against disorders of the bowel. However, the acid sensitivity of these cells results in a loss of viability during gastric passage and, consequently, a loss of efficacy. Probiotic treatment can be supplemented using ‘prebiotics’, which are carbohydrates fermented specifically by probiotic cells in the body. This combination of probiotic and prebiotic is termed a ‘synbiotic’. Within this article a multiparticulate dosage form has been developed, consisting of poly(d,l-lactic-co-glycolic acid) (PLGA) microcapsules containing prebiotic Bimuno™ incorporated into an alginate–chitosan matrix containing probiotic Bifidobacterium breve. The aim of this multiparticulate was that, in vivo, the probiotic would be protected against gastric acid and the release of the prebiotic would occur in the distal colon. After microscopic investigation, this synbiotic multiparticulate was shown to control the release of the prebiotic during in vitro gastrointestinal transit, with the release of galacto-oligosaccharides (GOS) initially occurred over 6 h, but with a triphasic release pattern giving further release over 288 h. Encapsulation of B. breve in multiparticulates resulted in a survival of 8.0 ± 0.3 log CFU/mL cells in acid, an improvement over alginate–chitosan microencapsulation of 1.4 log CFU/mL. This was attributed to increased hydrophobicity by the incorporation of PLGA particles.

Purification of coronavirus virions for Cryo-EM and proteomic analysis

Purification of intact enveloped virus particles can be useful as a first step in understanding the structure and function of both viral and host proteins that are incorporated into the virion. Purified preparations of virions can be used to address these questions using techniques such as mass spectrometry proteomics. Recent studies on the proteome of coronavirus virions have shown that in addition to the structural proteins, accessory and non-structural virus proteins and a wide variety of host cell proteins associate with virus particles. To further study the presence of virion proteins, high quality sample preparation is crucial to ensure reproducible analysis by the wide variety of methods available for proteomic analysis.

Production and purification of xylooligosaccharides from oil palm empty fruit bunch fibre by a non-isothermal process

Oil palm empty fruit bunches (OPEFB) fibre, a by-product generated from non-woody, tropical perennial oil palm crop was evaluated for xylooligosaccharides (XOS) production. Samples of OPEFB fibre were subjected to non-isothermal autohydrolysis treatment using a temperature range from 150 to 220 °C. The highest XOS concentration, 17.6 g/L which relayed from solubilisation of 63 g/100 g xylan was achieved at 210 °C and there was a minimum amount of xylose and furfural being produced. The chromatographic purification which was undertaken to purify the oligosaccharide-rich liquor resulted in a product with 74–78% purity, of which 83–85% was XOS with degree of polymerisation (DP) between 5 and 40.