Development and Characterization of Pectinate Micro/Nanoparticles for Gene Delivery

The aim of this study was to investigate the possibility of using pectinate micro/nanoparticles as gene delivery systems. Pectinate micro/nanoparticles were produced by ionotropic gelation. Various factors were studied for their effects on the preparation of pectinate micro/nanoparticles: the pH of the pectin solution, the ratio of pectin to the cation, the concentration of pectin and the cation, and the type of cation (calcium ions, magnesium ions and manganese ions). After the preparation, the size and charge of the pectin micro/nanoparticles and their DNA incorporation efficiency were evaluated. The results showed that the particle sizes decreased with the decreased concentrations of pectin and cation. The type of cations affected the particle size. Sizes of calcium pectinate particles were larger than those of magnesium pectinate and manganese pectinate particles. The DNA loading efficiency showed that Ca-pectinate nanoparticles could entrap DNA up to 0.05 mg when the weight ratio of pectin:CaCl₂:DNA was 0.2:1:0.05. However, Mg-pectinate could entrap only 0.01 mg DNA when the weight ratio of pectin:MgCl₂:DNA was 1:100:0.01 The transfection efficiency of both Ca-pectinate and Mg-pectinate nanoparticles yielded relatively low levels of green fluorescent protein expression and low cytotoxicity in Huh7 cells. Given the negligible cytotoxic effects, these pectinate micro/nanoparticles can be considered as potential candidates for use as safe gene delivery carriers.     

Colon-Targeted Delivery of IgY Against <Emphasis Type="Italic">Clostridium difficile</Emphasis> Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl₂ 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).     

Development of polysaccharide gel-coated pellets for oral administration: swelling and release behavior of calcium pectinate gel

The aim of this study was to examine the effect of pellet size, pectin type, pectin concentration, and dissolution medium on the swelling and drug release behavior of spherical pellets containing theophylline and coated with 2 different calcium pectinates, using a multi-level factorial design approach. The spherical pellets were prepared by an extrusion-spheronization method and then coated with calcium pectinate using the diffusion-controlled interfacial complexation technique, which provides a defect-free and uniform coating on solid cores. Theophylline release from the pellets was slowed by the application of the coatings. The time to release 50% of the payload (ie, T50) in an acidic medium was approximately 7 minutes from uncoated small pellets and was 55 minutes after an amidated calcium pectinate coat was applied; a comparable coat on large pellets showed a T50 of 93 minutes. Drug release profiles of dry coated pellets showed a lag time (all less than 20 minutes) when the gel coat hydrated and swelled, followed by a zero-order release. It was found that the release rate was controlled by the pellet size, pectin type, pectin concentration, and dissolution medium.     

Effect of polymeric cosolutes on calcium pectinate gelation. Part 3. Gum arabic and overview

Addition of gum arabic (average M_r≈450 kDa; 0.5–2.0 wt%) to solutions of low methoxy pectin (DE 31; 2.0 wt%; pH≈2.9–3.0) with stoichiometric Ca²⁺ caused massive increases in G′ and G″ in the pre-gel state at 90 °C (attributed to segregative interactions promoting formation of calcium-mediated ‘egg-box’ junctions between pectin chains) but had little effect on the gels formed on cooling to 5 °C. This is in marked contrast to the behaviour of other polymeric cosolutes studied in the investigations reported in the two preceding papers, which caused large reductions in gel moduli (attributed to excessive association of calcium pectinate into large aggregated bundles); the difference is tentatively ascribed to strengthening of the calcium pectinate network by divalent counterions to the uronate residues in gum arabic. When the complication of cation exchange was eliminated by extensive dialysis of gum arabic against 100 mM Na⁺ and use of the final dialysate in preparation of mixtures with calcium pectinate, massive increases in G′ and G″ at high temperature were again observed, but with accompanying reductions in moduli at low temperature, which, at gum arabic concentrations above 1.0 wt%, arose from collapse of the developing calcium pectinate network during cooling. The tentative conclusion from this work, and from the two preceding papers, is that enthalpically unfavourable (segregative) interactions between low methoxy pectin and polymeric cosolutes can be relieved in two ways: (i) Ca²⁺-mediated self-association of pectin into compact ordered assemblies which occupy less of the total volume, and (ii) conformational rearrangement of the cosolute molecules to minimise segmental interactions with pectin; conformational rearrangement is inhibited by chain stiffness and by branching; thus polymeric cosolute molecules of limited flexibility are more effective in promoting self-association of pectin than more flexible molecules of comparable size, and branched molecules are more effective than linear chains of comparable stiffness.     

Extraction, characterization and spontaneous gel-forming property of pectin from creeping fig (Ficus pumila Linn.) seeds

Creeping fig (Ficus pumila Linn.) seeds can be manually rubbed and squeezed to produce a water extract (WE) that can gel at room temperature without any additives. Its gelling material, extraction behavior, and mechanism of spontaneous gel-formation were investigated. Gelling material was extracted from seeds using water, ammonium oxalate and hydrochloric acid, respectively. Results showed the main component of three extracts is low methoxyl pectin. The pectin locates in a transparent layer on the surface of seeds, as revealed by an inverted microscope. Hence, explained the feasibility of the squeezing and rubbing method in traditional handcraft. Comparing with the other methods, water-extracted pectin has high galacturonic acid content, viscosity-average molecular weight and intrinsic viscosity but low degree of methoxylation. This pectin forms the major component of WE, together with the high amount of calcium ions present in WE, it suggests the spontaneous gelation may be based on the ‘egg-box’ formation.     

The influence of a specific microelemental environment in alginate gel beads on the course of propionic acid fermentation

In this work the exchange of calcium, cobalt, iron, magnesium, zinc and manganese ions between alginate gel beads and casein medium was investigated. The high release of calcium ions from alginate to the medium and the biosorption of some metal ions were observed. The pure alginate gel adsorbed all the metal ions examined, from a fermentative medium. Gel with immobilized cells of two strains of Propionibacterium freudenreichii subsp. shermanii showed an active ability to adsorb only cobalt, iron and zinc ions. In this way, a special microelemental environment was created in the alginate gel. This resulted in an increase of propionic acid production and a decrease of vitamin B₁₂ biosynthesis. Received: 30 April 1997 / Received revision: 2 July 1997 / Accepted: 4 July 1997     

Heat-induced gelation of bovine serum albumin/low-methoxyl pectin systems and the effect of calcium ions

Influence of low-methoxyl pectin (LM pectin) and calcium ions (3 mM) on mechanical behavior and microstructure of bovine serum albumin (BSA) gels (pH 6.8, in 0.1 M NaCl) was evaluated. Protein and LM pectin concentrations were fixed at 2, 4, and 8 wt % and 0.21, 0.43, and 0.85 wt %, respectively. Rheological measurements and confocal laser scanning microscopy coupled with texture image analysis by use of the co-occurrence method were performed. Heat treatment of BSA/LM pectin mixtures induced protein gelation and a phase separation process between the two biopolymers, which was kinetically trapped. Calcium ions induced pectin gelation and modified BSA gel properties. Depending on biopolymer concentrations, a balance between pectin and/or protein gel contribution on final gel strength exists. The microstructures of the mixed systems in the presence of calcium can be interpreted as interpenetrated structures. Texture image analysis allowed one to classify more precisely the different microstructures observed in relation with mechanical properties.     

Molecular dynamics simulation of the atomistic monolayer structures of N-acyl amino acid-based surfactants

Abstract

Atomic molecular dynamics simulations have been performed on the monolayer systems of N-acyl amino acid-based surfactants. The role of intermolecular hydrogen bonds and ionic side chain length of dicarboxylate surfactants were investigated through radial and spatial distribution functions. It was found that the hydrogen bonding capability between surfactants was the major factor determining the surface area each surfactant could occupy. Tighter packing of surfactants would lead to a weaker interaction with water molecule, and the protonation of carboxylate groups resulted in stronger inter-surfactant interactions. The hydrogen bonds with water molecules were found to prevail between the carboxylate groups, and regular cage-like water distributions surrounding the surfactant headgroups were seen. The introduction of divalent ions leads to a significant increase of counterion binding, and their intramolecular and intermolecular bindings of calcium ions were also well characterised. The intramolecular chelation of calcium ions was found impossible between the carboxylate groups for N-acyl glutamate due to its flexible side chain.     

FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.     

Pectin Matrix as Oral Drug Delivery Vehicle for Colon Cancer Treatment

Colon cancer is the fourth most common cancer globally with 639,000 deaths reported annually. Typical chemotherapy is provided by injection route to reduce tumor growth and metastasis. Recent research investigates the oral delivery profiles of chemotherapeutic agents. In comparison to injection, oral administration of drugs in the form of a colon-specific delivery system is expected to increase drug bioavailability at target site, reduce drug dose and systemic adverse effects. Pectin is suitable for use as colon-specific drug delivery vehicle as it is selectively digested by colonic microflora to release drug with minimal degradation in upper gastrointestinal tract. The present review examines the physicochemical attributes of formulation needed to retard drug release of pectin matrix prior to its arrival at colon, and evaluate the therapeutic value of pectin matrix in association with colon cancer. The review suggests that multi-particulate calcium pectinate matrix is an ideal carrier to orally deliver drugs for site-specific treatment of colon cancer as (1) crosslinking of pectin by calcium ions in a matrix negates drug release in upper gastrointestinal tract, (2) multi-particulate carrier has a slower transit and a higher contact time for drug action in colon than single-unit dosage form, and (3) both pectin and calcium have an indication to reduce the severity of colon cancer from the implication of diet and molecular biology studies. Pectin matrix demonstrates dual advantages as drug carrier and therapeutic for use in treatment of colon cancer.     

Mechanism of Biosorption of Heavy Metals by Mucor rouxii

Fungi such as Aspergillus niger and Mucor rouxii are capable of removing heavy metals from aqueous solutions. The role various functional groups play in the cell wall of M. rouxii in metal biosorption of lead, cadmium, nickel and zinc was investigated in this paper. The biomass was chemically treated to modify the functional carboxyl, amino and phosphate groups. These modifications were examined by means of infrared spectroscopy. It was found that an esterification of the carboxyl groups and phosphate and a methylation of the amine groups significantly decreased the biosorption of the heavy metals studied. Thus, the carboxylate, amine and phosphate groups were recognized as important in the biosorption of metal ions by M. rouxii biomass. The role the lipids fraction play was not significant. The study showed that Na, K, Ca and Mg ions were released from the biomass after biosorption of Pb, Cd, Ni and Zn, indicating that ion exchange was a key mechanism in the biosorption of metal ions by M. rouxii biomass.     

Studies on the growth of Saccharomycopsis fibuliger on pectic materials

Growth experiments have indicated that the yeast Saccharomycopsis fibuliger is able to utilize pectic materials as a carbon source for cell growth. Small quantities of yeast extract or mycological peptone and maltose are necessary to initiate growth. Cell yields of 35 g/100 g pectin were obtained after modification of the pH value, the concentration of calcium ions and pectin in the growth medium. The optimum pH for cell growth was 4.8, which is similar to the reported optimum for cell growth on starch. Cell yields declined at higher concentrations of pectin, as a result of the reduced rate of oxygen absorption into the growth medium. Neither the concentration of ammonium and phosphate ions nor the type of inorganic nitrogen source significantly affected cell growth within the experimental conditions employed.     

Di- and trinuclear arrangements of zinc(II)-1,5,9-triazacyclododecane units on the calix[4]arene scaffold: Efficiency and substrate selectivity in the catalysis of ester cleavage

The catalytic activity of the zinc(II) complexes of calix[4]arenes decorated with 1,5,9-triazacyclododecane ligands at the 1,2-, 1,3-, and 1,2,3-positions of the upper rim was investigated in the basic methanolysis (pH 10.4) of aryl acetates functionalised at the meta- and para-positions with a carboxylate anchoring group. Michaelis-Menten kinetics and turnover catalysis were observed. High rate accelerations, up to more than 10⁴-fold at 0.2 mM catalyst, were recorded in the most favourable catalyst-substrate combinations. The order of catalytic efficiency of regioisomeric bimetallic complexes is 1,2-vicinal ? 1,3-distal, resulting from a significant degree of synergism between metal ions in the former, and a complete lack in the latter. The moderately higher efficiency of the trimetallic compared with the 1,2-vicinal bimetallic catalyst provides an indication of a possible cooperation of three zinc(II) ions in the catalysis.     

Rheological behaviour and microstructure of microfibrillated cellulose suspensions/low-methoxyl pectin mixed systems. Effect of calcium ions

In this study, the influence of the presence of low-methoxyl pectin (LM pectin) on the rheological and microstructural properties of microfibrillated cellulose suspensions was elucidated in order to create new structures with new and interesting textures. For that purpose, the rheological properties of the cellulose/LM pectin mixtures in variable proportions were compared with those of the individual biopolymers. The influence of the presence of calcium and/or sodium ions on the properties of the mixed systems was studied. The microstructure of the resulting system was studied by transmission electron microscopy and confocal laser scanning microscopy. It was found that, in the presence of LM pectin, a synergistic effect was observed when calcium ions were also present, leading to increased rheological properties of the composites. Indeed, addition of calcium to the mixtures induced LM pectin gelation, which was favoured in the presence of sodium, the pectin network contributing to the formation of a stronger cellulose/LM pectin composite gel. The presence of LM pectin alone in the microfibrillated cellulose suspensions does not significantly modify the viscoelastic and microstructural properties of microfibrillated cellulose suspensions. Whether calcium was added to the mixtures or not in water, the viscoelastic properties of the mixtures are mainly controlled by cellulose. The same behaviour was observed for the mixtures in NaCl without added calcium. Contrary to this observation, it was noticed that in presence of both sodium and calcium ions, the viscoelastic properties of the mixtures are largely governed by LM pectin. On the other hand, it was showed that the flow behaviour of microfibrillated cellulose suspensions is modified in the presence of LM pectin with an increase in thixotropic character shear-thinning behaviour, which was more pronounced in the presence of NaCl. It was also shown, from TEM observations, that an interpenetrating network formed in cellulose/LM pectin composites gel in the presence of calcium ions. In the same way, the CLSM observations allowed the separate localization of cellulose and LM pectin within the composite systems to be highlighted. The results obtained suggests that it is possible to thus create new structures with new interesting textures, by mixing microfibrillated cellulose suspensions and LM pectin in suitable proportions in the simultaneous presence of both sodium and calcium ions.     

Calcium,magnesium and zinc ion coordination equilibria of vincristine

The zinc ion coordination of vincristine was studied by polarography; the analogous calcium ion coordination process was studied potentiometrically by a calcium ion selective electrode. In both cases, complexes of 1:1 composition were formed. The formation constant of the calcium complex was found to be 1g K = 3.27 ± 0.1. On the basis of the substitution of zinc in its vincristine complex by calcium and magnesium ions respectively, the ratio of the corresponding stability constants could be estimated as K_Zn:K_Ca (and K_Zn:K_Mg) ∼ 10⁵−3 × 10⁴. The complex formation processes proved to be pH-independent in the pH range 3.4–5.5, indicating that the metal ions are coordinated by the unprotonated oxygen donor atoms of vincristine.     

Structural insight into substrate binding and catalysis of a novel 2-keto-3-deoxy-D-arabinonate dehydratase illustrates common mechanistic features of the FAH superfamily

The archaeon Sulfolobus solfataricus converts d-arabinose to 2-oxoglutarate by an enzyme set consisting of two dehydrogenases and two dehydratases. The third step of the pathway is catalyzed by a novel 2-keto-3-deoxy-d-arabinonate dehydratase (KdaD). In this study, the crystal structure of the enzyme has been solved to 2.1 Å resolution. The enzyme forms an oval-shaped ring of four subunits, each consisting of an N-terminal domain with a four-stranded β-sheet flanked by two α-helices, and a C-terminal catalytic domain with a fumarylacetoacetate hydrolase (FAH) fold. Crystal structures of complexes of the enzyme with magnesium or calcium ions and either a substrate analog 2-oxobutyrate, or the aldehyde enzyme product 2,5-dioxopentanoate revealed that the divalent metal ion in the active site is coordinated octahedrally by three conserved carboxylate residues, a water molecule, and both the carboxylate and the oxo groups of the substrate molecule. An enzymatic mechanism for base-catalyzed dehydration is proposed on the basis of the binding mode of the substrate to the metal ion, which suggests that the enzyme enhances the acidity of the protons α to the carbonyl group, facilitating their abstraction by glutamate 114. A comprehensive structural comparison of members of the FAH superfamily is presented and their evolution is discussed, providing a basis for functional investigations of this largely unexplored protein superfamily.     

Role of Pectinesterase in pH-Dependent Interactions between Pea Cell Wall Polymers

Extracts of etiolated pea (Pisum sativum L.) shoots converted soluble pectin from the seedlings to a trichloroacetic acid-insoluble form. This activity coincided with pectinesterase peaks separated from the extracts by gel filtration and ion exchange. The conversion of pectin to the trichloroacetic acid-insoluble form and pectinesterase exhibited identical responses to pH, with activity only above pH 6. The formation of trichloroacetic acid-insoluble pectin in pea cell walls and their extracts is due to de-esterification of the pectin by pectinesterase and not to binding between pectin and a protein, as reported in the literature.     

Influence of zinc on the calcium carbonate biomineralization of Halomonas halophila

Background

The salt tolerance of halophilic bacteria make them promising candidates for technical applications, like isolation of salt tolerant enzymes or remediation of contaminated saline soils and waters. Furthermore, some halophilic bacteria synthesize inorganic solids resulting in organic–inorganic hybrids. This process is known as biomineralization, which is induced and/or controlled by the organism. The adaption of the soft and eco-friendly reaction conditions of this formation process to technical syntheses of inorganic nano materials is desirable. In addition, environmental contaminations can be entrapped in biomineralization products which facilitate the subsequent removal from waste waters. The moderately halophilic bacteria Halomonas halophila mineralize calcium carbonate in the calcite polymorph. The biomineralization process was investigated in the presence of zinc ions as a toxic model contaminant. In particular, the time course of the mineralization process and the influence of zinc on the mineralized inorganic materials have been focused in this study.

Results

H. halophila can adapt to zinc contaminated medium, maintaining the ability for biomineralization of calcium carbonate. Adapted cultures show only a low influence of zinc on the growth rate. In the time course of cultivation, zinc ions accumulated on the bacterial surface while the medium depleted in the zinc contamination. Intracellular zinc concentrations were below the detection limit, suggesting that zinc was mainly bound extracellular. Zinc ions influence the biomineralization process. In the presence of zinc, the polymorphs monohydrocalcite and vaterite were mineralized, instead of calcite which is synthesized in zinc-free medium.

Conclusions

We have demonstrated that the bacterial mineralization process can be influenced by zinc ions resulting in the modification of the synthesized calcium carbonate polymorph. In addition, the shape of the mineralized inorganic material is chancing through the presence of zinc ions. Furthermore, the moderately halophilic bacterium H. halophila can be applied for the decontamination of zinc from aqueous solutions.     

Effect of Calcium and Zinc on the Activity and Thermostability of Superoxide Dismutase

The effect of calcium and zinc ions on superoxide dismutase (SOD) from four plant species (Taxus baccata, Pinus sylvestris, Medicago rigidula, and Zea mays) was followed at three temperatures: optimal (20 °C), increased (50 °C), and high, inhibiting temperature (70 – 80 °C). At 20 and 50 °C in vitro added calcium increases SOD activity, but the degree was different for the plants investigated. The effect of zinc ions at the same temperatures varied in the investigated plants from activation to inhibition. An inhibiting effect of high temperature on SOD activity was diminished in the presence of calcium or zinc ions. It was shown that calcium and zinc ions can increase activity and thermostabilize different SOD isoforms.     

Purification and Properties of a Pectate Lyase in Erwinia aroideae

A strain of Erwinia aroideae produced an extracellular pectolytic enzyme under growth conditions with pectin or pectic acid as the inducer. This strain also produced a pectin lyase when nalidixic acid is added to a culture medium. The pectolytic enzyme produced under the growth conditions was purified approximately 40-fold from the culture fluid by carboxy- methyl cellulose and Sephadex G-75 gel column chromatographies. The purified enzyme was almost homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis, having a molecular weight of about 36,000 to 38,000. This enzyme, with optimal activity at pH 9.0 to 9.2, produced reaction products which had a strong absorption at 230 nm indicating a lyase type of the reaction. The enzyme activity was markedly stimulated by calcium ion and completely inhibited by cobalt and mercuric ions and by ethylenediaminetetraacetate. Pectic acid or pectin with lower methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectin with higher methoxyl content was used as a substrate. It was concluded that the enzyme produced under the normal growth conditions is an endo-pectate lyase and differs from the pectin lyase induced by nalidixic acid.