Microencapsulation of esterified krill oil, using complex coacervation.

The microencapsulation of the esterified krill oil (EKO), obtained from the transesterification of krill oil (KO) with 3,4-dihydroxyphenylacetic acid (DHPA), via complex coacervation and was investigated. The experimental findings showed that the DHPA and phenolic lipids (PLs) in the EKO affected the stability of the gelatine (GE)-EKO emulsion. To improve its stability, the effects of varying the pH of GE and the use of two emulsification devices, including the homogeniser and ultrasonic liquid processor were investigated, where the ultrasonic liquid processor was found to be a relatively more appropriate emulsification device. In addition, the capsules prepared using a pH of GE of 8.0 showed superior storage and had significantly (p <0.05) lower peroxide value as compared to those prepared with a pH of GE of 6.5. The microencapsulation of the EKO was effective in delaying the development of oxidation products during a period of 25 d of storage, at 25 °C.

Release of insulin from PLGA-alginate dressing stimulates regenerative healing of burn wounds in rats.

Burn wound healing involves a complex set of overlapping processes in an environment conducive to ischaemia, inflammation and infection costing $7.5 billion/year in the U.S.A. alone, in addition to the morbidity and mortality that occur when the burns are extensive. We previously showed that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization and stimulates angiogenesis. More recently, we developed an alginate sponge dressing (ASD) containing insulin encapsulated in PLGA [poly(D,L-lactic-co-glycolic acid)] microparticles that provides a sustained release of bioactive insulin for >20 days in a moist and protective environment. We hypothesized that insulin-containing ASD accelerates burn healing and stimulates a more regenerative, less scarring healing. Using heat-induced burn injury in rats, we show that burns treated with dressings containing 0.04 mg insulin/cm(2) every 3 days for 9 days have faster closure, a higher rate of disintegration of dead tissue and decreased oxidative stress. In addition, in insulin-treated wounds, the pattern of neutrophil inflammatory response suggests faster clearing of the burned dead tissue. We also observe faster resolution of the pro-inflammatory macrophages. We also found that insulin stimulates collagen deposition and maturation with the fibres organized more like a basket weave (normal skin) than aligned and cross-linked (scar tissue). In summary, application of ASD-containing insulin-loaded PLGA particles on burns every 3 days stimulates faster and more regenerative healing. These results suggest insulin as a potential therapeutic agent in burn healing and, because of its long history of safe use in humans, insulin could become one of the treatments of choice when repair and regeneration are critical for proper tissue function.

Polymerization Induced Self-Assembly of Alginate Based Amphiphilic Graft Copolymers Synthesized by Single Electron Transfer Living Radical Polymerization.

Alginate-based amphiphilic graft copolymers were synthesized by single electron transfer living radical polymerization (SET-LRP), forming stable micelles during polymerization induced self-assembly (PISA). First, alginate macroinitiator was prepared by partial depolymerization of native alginate, solubility modification and attachment of initiator. Depolymerized low molecular weight alginate (∼12 000 g/mol) was modified with tetrabutylammonium, enabling miscibility in anhydrous organic solvents, followed by initiator attachment via esterification yielding a macroinitiator with a degree of substitution of 0.02, or 1-2 initiator groups per alginate chain. Then, methyl methacrylate was polymerized from the alginate macroinitiator in mixtures of water and methanol, forming poly(methyl methacrylate) grafts, prior to self-assembly, of ∼75 000 g/mol and polydispersity of 1.2. PISA of the amphiphilic graft-copolymer resulted in the formation of micelles with diameters of 50-300 nm characterized by light scattering and electron microscopy. As the first reported case of LRP from alginate, this work introduces a synthetic route to a preparation of alginate-based hybrid polymers with a precise macromolecular architecture and desired functionalities. The intended application is the preparation of micelles for drug delivery; however, LRP from alginate can also be applied in the field of biomaterials to the improvement of alginate-based hydrogel systems such as nano- and microhydrogel particles, islet encapsulation materials, hydrogel implants, and topical applications. Such modified alginates can also improve the function and application of native alginates in food and agricultural applications.

Microencapsulation by interfacial polymerisation: membrane formation and structure.

Interfacial polymerisation was mainly developed toward the end of the 1960s, leading to applications in microcapsule production by the mid-1970s. The process consists in the dispersion of one phase containing a reactive monomer, into a second immiscible phase to which is added a second monomer. Both monomers react at the droplet surface (interface), forming a polymeric membrane. Over the last 50 years, many studies have been reported, but very few have provided a comprehensive review of this technology. This contribution reviews microcapsule production by interfacial polymerisation from the chemical, physico-chemical and physical perspectives, providing a tool for understanding and mastering this production technology, but also providing guidance toward improvements for future process design.

Microencapsulation of krill oil using complex coacervation.

The research work was aimed at the development of a process to yield gelatin-gum Arabic multinuclear microcapsules of krill oil (KO), via complex coacervation. On the basis of the experimental results of the screening trials, a three-level-by-three-factor Box-Behnken design was used to evaluate the effects of the ratio of the core material to the wall (RCW; x1), the stirring speed (SP; x2) and the pH (x3) on the encapsulation efficiency (EE). The experimental findings indicated that x3 has the most significant linear and quadratic effects on the EE of KO and a bilinear effect with x1, whereas x2 did not have any significant effect. The optimal conditions for a 92% of EE were: 1.75:1 for RCW, 3.8 for pH and 3 for SP. The microcapsules, formed by complex coacervation and without any cross-linking agent, were multinucleated, circular in shape and had sufficient stability to maintain their structure.

Characterization of biologically active insulin-loaded alginate microparticles prepared by spray drying.

BACKGROUND: Spray drying has been used as a means to encapsulate therapeutics in polymeric matrices to improve stability and alter pharmacokinetics. This research aims to characterize alginate microparticles formed by spray drying to encapsulate insulin for therapeutic delivery applications. METHODS: Particle size was characterized by laser diffraction spectroscopy, morphology by scanning electron microscopy, and protein and polymer distribution by confocal laser scanning microscopy. In addition, particle fines collected from the spray-dryer exhaust unit were characterized for size and morphology. The insulin encapsulation efficiency (EE) was determined after particle dissolution through quantification by spectrophotometric analysis. An in-vitro bioassay involving stimulation of rat L6 myoblasts was developed to confirm the bioactivity of released insulin. RESULTS: Mean diameter of the product was 2.1 ± 0.3 µm. Larger particles appeared spherical, with some smaller particles presenting surface topography variability and divoting. Protein EE was 38.2% ± 9.5%, with confocal microscopy showing the protein and polymer concentrated at the surface of larger particles, but more evenly distributed throughout smaller particles. A bioassay for the in-vitro quantification of insulin bioactivity was developed by calibrating the ratio of phosphorylated to total cellular protein kinase B (PKB; also known as AKT). in insulin-stimulated rat L6 myoblasts. Insulin released from the particles was 88% ± 15% bioactive, showing that spray drying had minimal impact on protein structure. CONCLUSION: Spray drying was effective in producing microparticles containing bioactive insulin. Future studies will focus on the improvement of the EE and particle uniformity with the aim of developing this technology further for the encapsulation and delivery of peptide or protein-based therapeutics.

Alginate-PEG sponge architecture and role in the design of insulin release dressings.

Wound healing is a natural process involving several signaling molecules and cell types over a significant period of time. Although current dressings help to protect the wound from debris or infection, they do little in accelerating the healing process. Insulin has been shown to stimulate the healing of damaged skin. We have developed an alginate sponge dressing (ASD) that forms a hydrogel capable of providing a moist and protective healing environment. By incorporating insulin-loaded poly(d,l-lactide-co-glycolide) (PLGA) microparticles into ASD, we successfully stabilized and released insulin for up to 21 days. Insulin release and water absorption and transfer through the ASD were influenced by altering the levels of poly(ethylene glycol) (PEG) in the dressing matrix. Bioactivity of released insulin can be maintained for at least 10 days, demonstrated using a human keratinocyte migration assay. Results showed that insulin-loaded PLGA microparticles, embedded within PEG-ASD, functioned as an effective long-term delivery platform for bioactive insulin.

Evaluation of hepatic glucose metabolism via gluconeogenesis and glycogenolysis after oral administration of insulin nanoparticles.

Nanoparticles were designed to promote insulin intestinal absorption via the oral route, to increase portal insulin levels to better mimic the physiological pathway, providing enhanced glucose control through glycogenolysis and gluconeogenesis. Nanoparticles were formulated with insulin encapsulated in the core material consisting of alginate and dextran sulfate, associated with poloxamer and subsequently coated with chitosan then albumin. A spherical and slightly rough core was observed in electron micrographs with the appearance of a concentration gradient of the polysaccharide structure toward the periphery of the nanoparticle. Atomic force microscopy showed that the fully formed nanoparticles are about 200 nm in diameter with smooth and spherical morphology. Histopathological analysis of organs and tissues of diabetic rats dosed daily for 15 days with insulin nanoparticles was used to evaluate toxicological issues. No morphological or pathological alterations were observed in rat liver, spleen, pancreas, kidney or intestinal sections. Following, the effect of nanoencapsulated insulin on inhibiting hepatic gluconeogenesis was evaluated after a single insulin administration and oral glucose tolerance test, which represents a significant metabolic challenge to the liver. Alterations in the hepatic glucose metabolism of fasted streptozotocin-diabetic rats were determined by the percent contribution of glycogenolysis and gluconeogenesis, measured by using metabolic tracers, however similar gluconeogenesis contribution to the hepatic metabolism was observed between diabetic rats receiving nanoencapsulated insulin or insulin solution. The metabolic results may be explained by the inability of a single dose in shifting the gluconeogenesis/glycogenolysis contributions, sampling time, fasting period or by influence of the kidney enzymes and impairment in insulin signaling observed in stz-diabetic rats.

Effects of an oral insulin nanoparticle administration on hepatic glucose metabolism assessed by ¹³C and ²H isotopomer analysis.

The purpose of this study was to evaluate hepatic glucose metabolism of diabetic induced rats after a daily oral load of insulin nanoparticles over 2 weeks. After the 2-week treatment, an oral glucose tolerance test was performed with [U-¹³C] glucose and ²H2O. Plasma glucose ²H and ¹³C enrichments were quantified and the contribution of glycogenolysis and gluconeogenesis to overall glucose production were estimated. Animals with the insulin nanoparticles displayed the lowest glycemia before the oral glucose tolerance test. In all animals, 75% of the total glucose production was from gluconeogenesis and glycogen synthesis was only detected in some animals. Gluconeogenic pathway was an active contributor to hepatic glucose production and the treatment with oral delivered insulin nanoparticles did not alter this contribution, suggesting that under this treatment, protocol hepatic glucose metabolism is not the most relevant target of insulin action but instead a more generalised effect in peripheral tissues.

Semisynthesis of a controlled stimuli-responsive alginate hydrogel.

Benefits of the use of natural polymers include biodegradability, biocompatibility, natural abundance, and unique physicochemical/biological properties. Native alginate was used to semisynthesize a new class of biomaterial in which the physical properties such as swelling and pore size can be chemically tailored for desired end use. Semisynthetic network alginate polymer (SNAP) was prepared by reaction with glutaraldehyde, forming an acetal-linked network polymer gel with carboxylate moieties preserved as stimuli-responsive sensors. The molecular structure of the hydrogel was confirmed by cross-polarization magic-angle spinning (13)C solid state NMR, and reaction parameters affecting the polymer synthesis, including reactant, catalyst concentrations, and solvent composition, were characterized by gel equilibrium swelling. The acetalization reaction can be thermodynamically controlled, offering fine-tuned control of gel swelling and pore properties. In addition, SNAP demonstrated pronounced swelling at alkaline pH and contraction in acidic environment with oscillatory response to repeated pH-stimuli, yielding a potential pulsatile, oral drug delivery vehicle. Through selection of reaction conditions, gel swelling, pore size, and stimuli-responsive characteristics can be specifically tailored for applications such as a tissue scaffold in regenerative medicine, as a targeted delivery vehicle, and as a superabsorbent in environmental cleanup.

Strategies toward the improved oral delivery of insulin nanoparticles via gastrointestinal uptake and translocation.

The design of strategies that improve the absorption of insulin through the gastrointestinal tract is a considerable challenge in the pharmaceutical sciences and would significantly enhance the treatment of diabetes mellitus. Several strategies have been devised to overcome physiologic and morphologic barriers to insulin absorption, including the inhibition of acidic and enzymatic degradation, enhancement of membrane permeability or widening of tight junctions, chemical modification of insulin, and the formulation of carrier systems. In particular, the concept of nanoparticulate carriers for oral insulin delivery has evolved through remarkable advances in nanotechnology. Investigations focused on uptake and translocation via Peyer's patches have demonstrated high levels of nanoparticle absorption based on significant alterations in the glycemic response to various glucogenic sources. This paper reviews the mechanisms for insulin and particle uptake and translocation through the gastrointestinal tract, and the potential barriers to this, outlines the design of nanoparticulate carriers for the oral delivery of insulin, and presents prospects for its clinical application.

Kinetic controlled synthesis of pH-responsive network alginate.

Alginates are of considerable interest in the fields of biotechnology and biomedical engineering. To enable the control of properties generally not possible with the native polymer, we have chemically modified alginate with dialdehyde via acid-catalyzed acetalization. The kinetics of acetalization measured through equilibrium swelling of the networked polymer were found to undergo a zero- and second-order reaction with respect to alginate and dialdehyde, respectively. With the determined rate constant of 19.06 microL x mole(-1) x s(-1) at 40 degrees C and activation energy of 78.58 kJ x mol(-1), a proposed predictive reaction model may be used a priori to select reaction conditions providing specific polymer properties. Gel swelling and average pore size were then able to be controlled between 80-1000-fold and 35-840 nm, respectively, by predictive estimation of reagent concentration and formulation conditions. This semisynthetic but natural polymer is stimuli-responsive exhibiting high water absorbency and may potentially be used as drug delivery vehicle for protein therapeutics.

Polyelectrolyte biomaterial interactions provide nanoparticulate carrier for oral insulin delivery.

Nanospheres are being developed for the oral delivery of peptide-based drugs such as insulin. Mucoadhesive, biodegradable, biocompatible, and acid-protective biomaterials are described using a combination of natural polyelectrolytes, with particles formulated through nanoemulsion dispersion followed by triggered in situ gel complexation. Biomaterials meeting these criteria include alginate, dextran, chitosan, and albumin in which alginate/dextran forms the core matrix complexed with chitosan and albumin coat. Smaller size and higher albumin-based acid-protective formulation was orally administered to diabetic rats and glucose reduction and physiological response analyzed. Insulin encapsulation efficiency was 90, 82, and 66% for uncoated, chitosan-coated, and albumin-chitosan-coated alginate nanospheres, respectively. The choice of coating polymer seems to influence insulin release profile and to be crucial to prevent peptic digestion. Physiological response following oral delivery showed that insulin albumin-chitosan-coated alginate nanospheres reduced glycemia approximately 72% of basal values. Albumin serves as an important enteric coating providing acid- and protease protection enabling uptake of active drug following oral dosage.

Oseltamivir phosphate released from injectable Pickering emulsions over an extended term disables human pancreatic cancer cell survival.

Pickering emulsions are colloidal dispersions stabilized by particles that either migrate to, or are formed at, the oil-water interface during emulsification. Here, we fabricated and characterized Pickering water-in-oil emulsions where molten glycerol monostearate crystallized at the surface of micron-sized water droplets and formed protective solid shells. We tested this emulsion as a reservoir delivery platform for the sustained release of low molecular weight hydrophilic molecules including sodium chloride (NaCl) and sodium citrate as model compounds, and the therapeutic oseltamivir phosphate (OP), the delivery of which was the ultimate goal of this research. The objective was to achieve long-term (30-day) release of challenging to encapsulate actives and ultimately demonstrate the sustained release of OP for 20-30 days from an injectable formulation. OP was used because of its anticancer properties targeting mammalian neuraminidase 1 (Neu1) involved in multistage tumorigenesis. All actives including OP encapsulated in Pickering emulsions displayed a near linear release profile over 30 days. It was demonstrated that the release could be modulated by the addition of a second, competing surfactant sorbitan monooleate, Span 80, to the emulsion at levels above its critical micelle concentration. OP released from the emulsions significantly reduced cell viability in the human PANC-1 pancreatic cancer cell line for up to 30 days. The findings from this study indicate a simple, potentially injectable formulation and method that is easily upscaled resulting in a stable product with the potential to fully retain small hydrophilic molecules/drugs for sustained, near linear release over days, weeks, and potentially months.

Sustained release of bioactive therapeutic proteins from a biodegradable elastomeric device.

Effective localized delivery of a therapeutic protein requires a biodegradable device capable of delivering active protein at a sustained rate, and at a concentration within its therapeutic window. The objective of this study was to demonstrate that a biodegradable elastomeric device can be made in a cylindrical geometry, and still retain the ability to release a variety of therapeutic proteins at a nearly constant rate in nanomolar concentration with high bioactivity. The elastomers were prepared with cylindrical geometry by photo-cross-linking an acrylated star-poly(epsilon-caprolactone-co-d,l-lactide) macromer. Vascular endothelial growth factor (VEGF), interferon-gamma (IFN-gamma), and interleukin-2 (IL-2) were co-lyophilized with excipients, then entrapped within the elastomer matrix by photo-polymerization. Under identical formulation conditions, these proteins were released at the same, nearly constant rate for a significant part of the release profile (until 70%-80% release depending on formulation characteristics). Decreasing the molecular weight of the acrylated macromer increased the rate of protein release, but did not alter the zero order nature of the release kinetics. Cell based bioactivity assays showed only that 57% of the VEGF released was bioactive. By contrast, both IL-2 and IFN-gamma showed relatively high bioactivity and over 80% of the released proteins were bioactive. The elastomer formulation has potential as a regio-specific protein delivery device.

Maintenance of vascular endothelial growth factor and potentially other therapeutic proteins bioactivity during a photo-initiated free radical cross-linking reaction forming biodegradable elastomers.

Previously, we prepared a biodegradable elastomeric device that can release different therapeutic proteins at a nearly constant rate in nanomolar concentrations with high bioactivity. The elastomer device was fabricated using a photo-initiated free radical cross-linking reaction of acrylated star(epsilon-caprolactone-co-D,L-lactide) in organic solvent in the presence of solid protein particles. The objective of this study was to examine the effect of various parameters used for fabricating the photo-cross-linked elastomeric device on the stability of a therapeutic protein, vascular endothelial growth factor (VEGF), to determine which factor plays the dominant role in protecting VEGF. VEGF was lyophilized with or without bovine serum albumin (BSA) and then suspended in solid state in a macromer (acrylated star-poly(epsilon-caprolactone-co-D,L-lactide)) solution containing different concentrations of a free radical initiator, 2,2-dimethoxy-2-phenylacetophenone (DMPA). The protein suspension was then UV-irradiated at different intensities. UV irradiation with the generation of free radicals was detrimental to VEGF stability. BSA preserved the VEGF bioactivity during UV irradiation but provided little protection in the presence of the photo-initiator DMPA. The acrylated macromer acted as a free radical scavenger and effectively preserved VEGF and BSA stability during UV-initiated photo-polymerization. The detrimental effect of UV radiation with free radical generation on VEGF stability during device manufacture can be eliminated by choosing the proper bulking agents coupled with an efficient photo-polymerization reaction.

Nanoparticulate delivery system for insulin: design, characterization and in vitro/in vivo bioactivity.

Insulin-loaded alginate-dextran nanospheres were prepared by nanoemulsion dispersion followed by triggered in situ gelation. Nanospheres were characterized for mean size and distribution by laser diffraction spectroscopy and for shape by transmission electron microscopy. Insulin encapsulation efficiency and in vitro release were determined by Bradford protein assay and bioactivity determined in vitro using a newly developed Western blot immunoassay and in vivo using Wistar diabetic rats. Nanospheres ranged from 267 nm to 2.76 microm in diameter and demonstrated a unimodal size distribution. Insulin encapsulation efficiency was 82.5%. Alginate-dextran particles suppressed insulin release in acidic media and promoted a sustained release at near neutral conditions. Nanoencapsulated insulin was bioactive, demonstrated through both in vivo and in vitro bioassays.

Optimization of chlorophyllase-catalyzed hydrolysis of chlorophyll in monophasic organic solvent media.

The effects of selected reaction parameters, including solvent hydrophobicity, initial water activity, agitation speed, temperature and enzyme concentration, on the biocatalytic efficiency of a chlorophyllase enzymatic extract from Phaeodactylum tricornutum in neat organic solvent media were investigated. The highest chlorophyllase specific activity of 322 nmol hydrolyzed chlorophyll per gram of protein per minute and bioconversion yield of 91% were obtained in the reaction mixture of hexane/2-octanone (98.3:1.7, v/v), at a controlled initial water activity of 0.90. R O/A value, which is the ratio of the specific activity in the organic solvent to that in the aqueous/miscible organic solvent medium, was 1.5 x 10-3. To reduce the substrate diffusional limitations, the appropriate agitation speed and enzyme concentration were determined. The optimum temperature for maximal enzymatic activity and activation energy were 35 degrees C and 105.0 kJ/mol, respectively. Although the catalytic efficiency of chlorphyllase in the neat organic solvent mixture was lower than that in the aqueous medium, its half-life time in the first environment at temperature ranging from 35 to 50 degrees C was increased by 5.0 to 15.0 times.

Irreversible fouling during multicycle microfiltration of wastewater effluent.

This study focused on irreversible fouling during microfiltration of primary and secondary effluents from municipal wastewater treatment plants. Flow resistances were calculated from the sum of clean membrane resistances, resultant cake layer resistances, and consequent irreversible fouling resistances. Results from a dead-end cell experimental system showed that the accumulated cake resistance was dominating for microfiltration of primary/secondary effluents. Suspended solids in the primary and secondary effluents had a similar compressibility index, n, with a value of approximately 0.5, indicating that they were moderately compressible particles. The value of irreversible resistance is dependent on the intensity of membrane cleaning; however, for a given membrane cleaning strategy, this value steadily increased and reached a maximum after approximately 6 cycles of filtration and cleaning. This study provided an explanation for the significant drop of throughput flux in the early application of membrane processes, and a plateau flux approached correspondingly.

Kinetics of chemoheterotrophic microbially mediated reduction of ferric EDTA and the nitrosyl adduct of ferrous EDTA for the treatment and regeneration of spent nitric oxide scrubber liquor.

Biomass from a prototype reactor was used to investigate the kinetics of chemoheterotrophic reduction of solutions of ferric ethylenediaminetetraacetic acid (EDTA) and solutions containing the nitrosyl adduct of ferrous EDTA using ethanol as the primary electron donor and carbon source. A series of batch experiments were conducted using biomass extracted from the scrubber solution treatment and regeneration stage of a prototype iron EDTA-based unit process for the absorption of nitric oxide with subsequent biological treatment. Using a linear-sweep voltammetric method for analysis of the ferric EDTA concentration, iron-reducing bacteria were found to behave according to the Monod kinetic model, at initial concentrations up to 2.16 g chemical oxygen demand (COD) as ethanol per liter, with a half-velocity constant of 0.532 g COD as ethanol/L and a maximum specific utilization rate of 0.127 mol/L of ferric ethylenediamine-tetraacetic acid [Fe(III)EDTA]*(g volatile suspended solids [VSS]/L)d(-1). Based on batch analyses, biomass yield and endogenous decay values of iron-reducing bacteria were estimated to be 0.055 g VSS/g COD and 0.017 L/d, respectively. An average of 1.64 times the theoretical (stoichiometric) demand of ethanol was used to complete reduction reactions. Kinetics of the reduction of the nitrosyl adduct of ferrous EDTA are summarized by the following kinetic constants: half-velocity constant (Ks) of 0.39 g COD/L, maximum specific utilization rate (k) of 0.2 mol/L [NO x Fe(II)EDTA(2-)](g VSS/L)d(-1), and inhibition constant (K(I)) of 0.33 g COD/L, as applied to the modified Monod kinetic expression described herein. Based on batch analyses, the biomass yield of nitrosyl-adduct-reducing bacteria was estimated to be 0.259 g VSS/g COD, endogenous decay was experimentally determined to be 0.0569 L/d, and an average of 1.26 times the stoichiometric demand of ethanol was used to complete reduction reactions.