Insulin adsorption on coated silica based supports grafted with N-acetylglucosamine by liquid affinity chromatography

Silica beads are coated with dextran carrying a calculated amount of positively charged diethylassminoethyl groups (DEAE) in order to neutralize negative charged silanol groups at the silica surface and in this way to minimize non specific interactions between silica surface and proteins in solution. Dextran-coated silica supports are potentially excellent stationary phases for high-performance liquid chromatography of proteins. These supports combine the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. These supports (silica-dextran-DEAE = SID) are easily functionalized by grafting N-acetylglucosamine (GlcNAc) using conventional coupling methods. The performances of the support bearing GlcNAc are studied by high-performance liquid affinity chromatography (HPLAC) of insulin, the hypoglycemic peptide hormone of the human organism. The study shows that these supports exhibit a reversible and specific affinity towards insulin and allow separations with high purification yields. Moreover, the influence of different physico-chemical parameters (pH, NaCl and insulin concentration) on insulin retention on the support was analysed. This allowed us to optimize the conditions of adsorption and to better understand the interaction mechanisms between insulin and GlcNAc as biospecific ligand.     

Interpenetrating biopolymer network based hydrogels for an effective drug delivery system

Discovery of hydrogels has resulted in developing competent controlled-release drug delivery systems. Present study describes the synthesis and characterization of novel pH responsive hydrogels of chitosan, hydroxyl ethyl cellulose (HEC) and polyol prepared by physical blending of the three components in different ratios. Vegetable oil derived polyol seems to act as a filler and cross linking agent. The synthesized hydrogels were characterized using FT-IR spectroscopy, thermo gravimetric analysis (TGA), Optical microscopy and scanning electron microscopy (SEM). Equilibrium swelling behavior of hydrogels in water and different buffers with pH values (2, 4, 7.3, and 8) indicated the sustained expansion of the films in different pH solutions.     

Targeted delivery of doxorubicin: Drug delivery system based on PAMAM dendrimers

Polyamidoamine (PAMAM) dendrimers of the second generation (G2) are branched polymers containing 16 surface amino groups that allow them to be used as universal carriers on creating systems for drug delivery. G2 labeled with fluorescein isothiocyanate (FITC) efficiently bound with the surface of tumor cells at 4°C and was absorbed by the cells at 37°C. The covalent binding to G2-FITC of a vector protein, a recombinant fragment of the human alpha-fetoprotein receptor-binding domain (rAFP3D), increased the binding and endocytosis efficiency more than threefold. Covalent conjugates of G2 with doxorubicin (Dox) obtained by acid-labile linking of cis-aconitic anhydride (CAA) without the vector protein (G2-Dox) and with the vector protein rAFP3D (rAFP3D-G2-Dox) were accumulated by the tumor cells with high efficiency. However, a selective effect was observed only in rAFP3D-G2-Dox, which also demonstrated high cytotoxic activity against the human ovarian adenocarcinoma SKOV3 cells and low cytotoxicity against human peripheral blood lymphocytes. Based on these results, rAFP3D-G2 conjugate is promising for selective delivery of antitumor drugs.     

Small molecule-based chemical affinity system for the purification of proteins

A new chemical affinity system is described for the purification of proteins. The Linx Affinity Purification System enables researchers to quickly and easily bind a protein ligand to a chromatographic matrix and use the resulting affinity resin to purify a second protein from crude mixtures. The entire process takes approximately 2 h.     

The rifampicin drug delivery system based on phospholipid nanoparticles

Low bioavailability of rifampicin, one of the main antituberculous agents, stimulates searches of its new optimized formulations. The present study has shown a possibility of rifampicin incorporation into nanoparticles from plant phosphatidylcholine (diameter of 20–30 nm). Addition of sodium oleate to the phospholipid system caused a 2-fold increase in the percent of rifampicin incorporation. The maximal concentration of rifampicin assayed in plasma samples by LC/MS was observed 1 h after oral administration to rats (6 mg/kg) and represented 0.5 and 4.2 μg/mL for free rifampicin and rifampicin incorporated in the phospholipids-oleate nanoparticles, respectively. These levels were maintained for more than 2 h of the experiment. High rifampicin bioavailability in the oleate containing phospholipid nanosystem suggests its prospects for practical use.     

An early warning system for aquatic environment state monitoring based on an analysis of mussel valve movements

An early warning system for biological monitoring of aquatic environment is proposed, which is based on an analysis of mussel valve movements. It consists of an analog monitor of an original design (analog recorder and analyzer of opening/closing of valves in a group of mussels) and outside devices (multimeters, computers, and mobile phones). In the monitor, an analysis of the state of valve position sensors, summing/averaging of voltage values, and generation of a warning signal are performed in the analog mode. The proposed system (a) diagnoses the state of valve position sensors, (b) determines the voltage representing the average valve opening in a group of mussels (6–12 individuals), (c) generates a warning signal; and, by means of peripheral devices, (d) registers the average voltage of sensors and (e) transmits a warning signal via a mobile communication net. The system can provide an addition (and, sometimes, an alternative) to complicated digital systems analogous to MosselMonitor®.     

The Dac-tag, an affinity tag based on penicillin-binding protein 5

Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some β-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the β-lactam ring. This process takes several minutes. We exploited this process and showed that a fragment of PBP5 can be used as a reversible and monomeric affinity tag. At ambient temperature (e.g., 22°C), a PBP5 fragment binds rapidly and specifically to ampicillin Sepharose. Release can be facilitated either by eluting with 10mM ampicillin or in a ligand-free manner by incubation in the cold (1-10°C) in the presence of 5% glycerol. The "Dac-tag", named with reference to the gene dacA, allows the isolation of remarkably pure fusion protein from a wide variety of expression systems, including (in particular) eukaryotic expression systems.     

Bifunctional monolithic affinity hydrogels for dual-protein delivery

Multiple-protein delivery has been proven to be a critical consideration for promoting tissue regeneration. Many polymeric composite biomaterials have been designed and used for modulating dual-protein delivery to enhance tissue regeneration in vitro or in vivo. However, the fabrication conditions and low water contents within the portions of these composite matrices that determine protein release rates are not optimal for maintaining the stability of encapsulated macromolecular therapeutics. In this proof-of-concept work, we aim to resolve this deficiency by single-step fabrication of affinity hydrogels capable of independently delivering two or more proteins. Selective protein-binding sites were incorporated into poly(ethylene glycol) hydrogels via copolymerization with glycidyl methacrylate-iminodiacetic acid (GMIDA) ligands to modulate release of two model proteins, lysozyme and hexahistidine tagged green fluorescent protein (hisGFP), via two distinct matrix-binding mechanisms, namely electrostatic interaction and metal-ion chelation. Differing from composite matrices for dual-protein delivery, the results reported herein indicate that injectable monolithic affinity hydrogels are capable of rapidly encapsulating multiple therapeutic agents under mild physiological conditions and independently controlling their localized delivery. Most importantly, these affinity hydrogels retain high water permeabilities throughout the entire device, characteristics that are necessary for maintaining the stability and viability of encapsulated proteins and cells.     

Development of a novel probiotic delivery system based on microencapsulation with protectants

The establishment of the health-promoting benefits of probiotics is challenged by the antimicrobial bio-barriers throughout the host’s gastrointestinal (GI) tract after oral administration. Although microencapsulation has been frequently utilised to enhance the delivery of probiotics, microcapsules of sub-100 μm were found to be ineffective and therefore questioned as an effective delivery vehicle for viable probiotics despite the sensory advantage. In this study, four probiotics strains were encapsulated in chitosan-coated alginate microcapsules of sub-100 μm. Only a minor protective effect was observed from this original type of microcapsule. In order to enhance the survival of these probiotics, sucrose, a metabolisable sugar, and lecithin vesicles were added to the wall material. Both of the ingredients could be readily encapsulated with the probiotics, and protected them from stresses in the simulated GI fluids. The metabolisable sugar effectively increased the survival of the probiotics in gastric acid, mainly through energizing the membrane-bound F₁F₀-ATPases. The lecithin vesicles proved to alleviate the bile salt stress, and hence notably reduced the viability loss at the elevated bile salt concentrations. Overall, three out of the total four probiotics in the reinforced sub-100 μm microencapsules could significantly survive through an 8-h sequential treatment of the simulated GI fluids, giving less than 1-log drop in viable count. The most vulnerable strain of bifidobacteria also yielded a viability increase of 3-logs from this protection. In conclusion, the sub-100 μm microcapsules can be a useful vehicle for the delivery of probiotics, as long as suitable protectants are incorporated in the wall matrix.     

Development of a microRNA delivery system based on bacteriophage MS2 virus-like particles

Recently, microRNA (miRNA)-mediated RNA interference has been developed as a useful tool in gene function analysis and gene therapy. A major obstacle in miRNA-mediated RNAi is cellular delivery, which requires an efficient and flexible delivery system. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for RNA and drug delivery. However, MS2 VLP-mediated miRNA delivery has not yet been reported. We therefore used an Escherichia coli expression system to produce the pre-miR 146a contained MS2 VLPs, and then conjugated these particles with HIV-1 Tat(47-57) peptide. The conjugated MS2 VLPs effectively transferred the packaged pre-miR146a RNA into various cells and tissues, with 0.92-14.76-fold higher expression of miR-146a in vitro and about two-fold higher expression in vivo, and subsequently suppressed its targeting gene. These findings suggest that MS2 VLPs can be used as a novel vehicle in miRNA delivery systems, and may have applications in gene therapy.     

Development of target delivery system based on actinomycin class drugs and recombinant alpha-fetoprotein

A recombinant alpha-fetoprotein (rAFP) was obtained in the yeast P. pastoris system, and its functional activity was confirmed. A method for producing polymer particles loaded with dactinomycin was developed, and a conjugate of these nanoparticles with rAFP was synthesized. The efficiency of the obtained conjugate on the HeLa, SKOV3, and MG-63 tumor cells and the absence of toxicity on the normal cells was shown. Experiments in vivo demonstrated a significant increase in the antitumor efficacy of the conjugate at a lower general toxicity as compared to the commercially available dactinomycin.     

Xenopus: an ideal system for chemical genetics

Chemical genetics, or chemical biology, has become an increasingly powerful method for studying biological processes. The main objective of chemical genetics is the identification and use of small molecules that act directly on proteins, allowing rapid and reversible control of activity. These compounds are extremely powerful tools for researchers, particularly in biological systems that are not amenable to genetic methods. In addition, identification of small molecule interactions is an important step in the drug discovery process. Increasingly, the African frog Xenopus is being used for chemical genetic approaches. Here, we highlight the advantages of Xenopus as a first-line in vivo model for chemical screening as well as for testing reverse engineering approaches.     

Electrochemical biosensing based on universal affinity biocomposite platforms

Rigid conducting biocomposites are versatile and effective transducing materials for the construction of a wide range of amperometric biosensors such as immunosensors, genosensors and enzymosensors, particularly if the transducer is bulk-modified with universal affinity biomolecules. The strept(avidin)-graphite-epoxy biocomposite could be considered as an universal immobilization platform whereon biotinylated DNAs, oligonucleotides, enzymes or antibodies can be captured by means of the highly affinity (strept)avidin-biotin reaction. Universal affinity biocomposite-based biosensors offer many potential advantages compared to more traditional electrochemical biosensors commonly based on a biologically surface-modified transducer. The integration of many materials into one matrix is their main advantage. As biological bulk-modified materials, the conducting biocomposites act not only as transducers, but also as reservoir for the biomaterial. After its use, the electrode surface can be renewed by a simple polishing procedure, establishing a clear advantage of these approaches relative to classical biosensors and other common biological assays. Moreover, the same material is useful for the analysis of many molecules whose determinations are based on genetic, enzymatic or immunological reactions. The different strategies for electrochemical genosensing, immunosensing and enzymosensing, all of them being dependent on the presence of a redox enzyme marker for the generation of the electrochemical signal, based on this universal affinity biocomposite platform are all presented and discussed.     

Bacterial ghosts are an efficient delivery system for DNA vaccines

Mass implementation of DNA vaccines is hindered by the requirement of high plasmid dosages and poor immunogenicity. We evaluated the capacity of Mannheimia haemolytica ghosts as delivery system for DNA vaccines. In vitro studies showed that bacterial ghosts loaded with a plasmid carrying the green fluorescent protein-encoding gene (pEGFP-N1) are efficiently taken up by APC, thereby leading to high transfection rates (52-60%). Vaccination studies demonstrated that ghost-mediated delivery by intradermal or i.m. route of a eukaryotic expression plasmid containing the gene coding for beta-galactosidase under the control of the CMV immediate early gene promoter (pCMVbeta) stimulates more efficient Ag-specific humoral and cellular (CD4(+) and CD8(+)) immune responses than naked DNA in BALB/c mice. The use of ghosts also allows modulating the major Th response from a mixed Th1/Th2 to a more dominant Th2 pattern. Intravenous immunization with dendritic cells loaded ex vivo with pCMVbeta-containing ghosts also resulted in the elicitation of beta-galactosidase-specific responses. This suggests that dendritic cells play an important role in the stimulation of immune responses when bacterial ghosts are used as a DNA delivery system. Bacterial ghosts not only target the DNA vaccine construct to APC, but also provide a strong danger signal, acting as natural adjuvants, thereby promoting efficient maturation and activation of dendritic cells. Thus, bacterial ghosts constitute a promising technology platform for the development of more efficient DNA vaccines.     

Studies on scale-up parameters of an immunoglobulin separation system using protein A affinity chromatography.

Effects of operational and system parameters on process scale-up of murine immunoglobulin (IgG2a kappa) purification using Protein A affinity chromatography are investigated. Parameters studied are those related to sample application, elution, ligand concentration on support, and column size change. Between sample application velocities of 0.004 and 0.030 cm/s (16-108 cm/h), the product concentration profiles in eluate did not show significant differences. With given system parameters, the retention time and bandwidth of the peak could be predicted by moment theory. The mean equilibrium constant during elution showed a strong effect of pH between 2 and 4. Within the range of protein A concentrations studied, 0.15-1.22 mg/mL of gel, the column capacity shows a linear relationship with the concentration of protein A immobilized. Dimensional scale-up of the column in the radial direction increased the total purification capacity linearly as the cross-sectional area increased without increasing pressure drop; however, the product concentration was diluted. Scale-up of the column dimension in the axial direction enables higher concentrations of product in the eluate, although the retention time increases linearly as the gel height increases.     

A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles

Delivery of DNA and siRNA into mammalian cells is a powerful technique in treating various diseases caused by single gene defects. Herein, we report a highly efficient delivery system using 1,4-butanediol diglycidyl ether (bisepoxide) crosslinked polyethylenimine (PEI) nanoparticles (PN). The nanoparticle/DNA complexes (nanoplexes) exibited approximately 2.5- to 5.0-fold gene transfer efficacy and decreased cytotoxicity in cultured cell lines, compared to the native PEI (25 kDa) (gold standard) and commercially available transfection agents such as Lipofectamine 2000 and Fugene. The bisepoxide crosslinking results in change in amine ratio in PEI; however, it retains the net charge on PN unaltered. A series of nanoparticles obtained by varying the degree of crosslinking was found to be in the size range of 69-77 nm and the zeta potential varying from +35 to 40 mV. The proposed system was also found to deliver siRNA efficiently into HEK cells, resulting in approximately 70% suppression of the targetted gene (GFP).     

Insulin 3: From chemical synthesis to biological function

Summary Insulin-like peptide 3 (INSL3) is one of ten members of the human insulin superfamily and consists of two peptide chains that contain the characteristic insulin fold and disulfide bond pairings. It is primarily produced in the Leydig cells of the testes, and gene knockout experiments have identified a key biological role as initiating testes descent during foetal development. Its receptor has recently been shown to be a member of the leucine-rich repeat-containing G-protein-coupled receptor family (LGR) and is known as LGR8. Considerable work has recently been undertaken with the aim of studying the mechanism of INSL3 downstream action on responsive cells and, towards this goal, the use of synthetic peptides has proved particularly beneficial. This mini-review outlines how these together with basic structure-function studies are beginning to reveal not only its molecular actions but also its potential new biological actions.     

Insulin substitution: new insulins, new modes of delivery

The demonstrated role of the tight control of hyperglycaemia for the prevention of long-term diabetic complications has reoriented the goals of insulin supply toward the search for restoration of the effects of physiological insulin secretion rather than the simple survival of insulin deficient patients and the reduction in the number of daily insulin injections to be performed. Normal blood glucose control requires the availability of a fast-acting insulin therapy at meal time in order to reduce hyperglycaemic excursions and a basal insulin therapy able to stabilize blood glucose between meals. Reduction of induced hypoglycaemic risk represents the secondary objective beside the main goal of avoiding hyperglycaemia. Fast-acting analogues, by a faster dissociation of their hexameric conformation after their injection or infusion in subcutaneous tissue, reduce post-meal hyperglycaemia, while their shortened duration of action versus regular insulin minimizes late post-absorptive risk of hypoglycaemia. Long-acting analogues, by their precipitation in subcutaneous tissue or their slowly reversible binding to albumin, provide a benefit on blood glucose stability versus NPH or zinc insulins. Continuous insulin therapy using pumps offers both a better blood glucose stability than multiple daily injections and a broader flexibility in life mode. Using the peritoneal route by implantable pumps is a mean to improve blood glucose stability in poorly controlled patients in spite of optimized subcutaneous insulin therapy. The development of glucose sensors provides reinforced information on blood glucose, versus self-monitoring by capillary blood measurements, that contributes to a better adaptation of insulin therapy. First trials of connections between blood glucose data and insulin delivery open a perspective toward glucose-modulated insulin therapy, at least in periods outside meals, leading to first models of semi-automated artificial endocrine pancreas. The alternative of a cellular insulin supply by pancreas or islet transplantation looked promising during recent years, but lack of transplants and adverse events related to immune suppression limit their use to very specific cases where benefit/risk ratio is positive.