Microencapsulation strategies for islet transplantation

A variety of islet microencapsulation techniques have been investigated to establish which method provides the least occlusive barrier to net insulin release in vitro, and optimum biocompatibility for islet implantation in vivo. NMRI mouse islets have been microencapsulated with Na+ -alginate-poly-L-lysine (PLL)/poly-L-ornithine (PLO)-alginate, Ba2+ -alginate and agarose gels. Both free and microencapsulated islets responded to glucose challenge in static incubation and perifusion by significantly increasing their rate of insulin release and theophylline significantly potentiated the insulin response to glucose. While little insulin was released from microencapsulated islets after short term (2 hours) static incubation, significantly greater amounts were released in response to glucose challenge after extended (8-24 hours) incubation. However, insulin release from all types of microencapsulated islets was significantly reduced compared with free islets. Na+ -alginate-PLO-alginate microencapsulated islets were significantly more responsive to elevated glucose than Na+ -alginate-PLL-alginate microencapsulated islets, due to the enhanced porosity of PLO membranes. The outer alginate layer created a significant barrier to glucose/insulin exchange and reduced the insulin responsiveness of microencapsulated islets to glucose. Ba2+ -alginate membrane coated islets, generated by the density gradient method, were the most responsive to glucose challenge. Low concentrations of NG-monomethyl L-arginine (L-NMMA) had no significant effect on glucose stimulated insulin release from either free or microencapsulated islets. However, 1.0 mmol/1 L-NMMA significantly inhibited the insulin response of both free and microencapsulated islets to glucose challenge. In vivo work designed to evaluate the extent of pericapsular fibrosis after 28 days ip. and sc. implantation of microencapsulated islets into STZ-diabetic recipients, revealed that the inclusion of islets within microcapsules increased their immunogenicity and markedly increased the extent of pericapsular fibrosis. When the outer alginate layer was omitted from microcapsules, little or no pericapsular mononuclear cell deposition was observed. The subcutaneous site was not suitable for microencapsulated islet transplantation in NMRI recipient mice. Systemic immunosuppression using cyclosporin A was effective in preventing pericapsular mononuclear cell deposition, while L-NMMA loading into microcapsules had no significant effect on pericapsular fibrosis, although it did maintain the integrity of microencapsulated islets.

3D culture of bone-derived cells immobilised in alginate following light-triggered gelation

Photoreactive liposomes have been exploited as a means of developing 3D tissue constructs. Liposomes formulated using the photosensitive lipid 1,2-bis(4-(n-butyl)phenylazo-4′-phenylbutyroyl)phosphatidylcholine (Bis Azo PC), which undergoes conformational change on stimulation with long wavelength ultraviolet light, were prepared with entrapped CaCl2 before being incorporated into a 4% alginate solution. It was shown that stimulation of the photosensitive lipid using a light emitting diode (LED) (peak emission at 385 nm, dose equivalent to 9 mJ/cm2) caused the release of liposome-entrapped CaCl2, resulting in cross-linking of the alginate solution and immobilisation of bone-derived cells over a range of seeding densities, approximately 97% of which remained viable for periods of up to 14 days in culture. Entrapment volumes of a variety of liposome types were evaluated and interdigitating fusion vesicles were identified as having the highest payload (24%), however the inclusion of cholesterol as a means of shifting Bis Azo PC sensitivity into the visible light wavelengths resulted in an approximately 10-fold reduction in calcium entrapment. This application of light-sensitised liposomes offers the potential to create complex tissue engineering substrates containing cells immobilised in precise locations, in contrast with substrates onto which cells are seeded post-production. © 2007 Elsevier B.V. All rights reserved.

Role of AmpR and alginate production in Pseudomonas aeruginosa biofilm antibiotic resistance associated with cystic fibrosis

Pseudomonas aeruginosa is an ubiquitous Gram-negative opportunistic pathogen that is commonly found in nosocomial infections, immunocompromised patients and burn victims. In addition, P. aeruginosa colonizes the lungs of cystic fibrosis patients, leading to chronic infection, which inevitably leads to their demise. In this research, I analyzed the factors contributing to P. aeruginosa antibiotic resistance, such as the biofilm mode of growth, alginate production, and 13-lactamase synthesis. Using the biofilm eradication assay (MBEC™ assay), I exposed P. aeruginosa to B-lactams (piperacillin, ceftazidime, and cefotaxime ), aminoglycosides ( amikacin, tobramycin and gentamicin), and a fluoroquinolone ( ciprofloxacin) at various concentrations. I analyzed the effects of biofilm on P. aeruginosa antibiotic resistance, and confirmed that the parent strain PAO 1 biofilms cells were > 100 times more resistant than planktonic (freefloating) cells. The constitutively alginate-producing strain PDO300 exhibited an altered resistance pattern as compared to the parent strain P AO 1. Finally, the role of AmpR, the regulator of ampC-encoded 13-lactamase expression was analyzed by determining the resistance of the strain carrying a mutation in the ampR gene and compared to the parent strain PAOl. It was confirmed that the loss of ampR contributes to increased antibiotic resistance.

Effect of experimental parameters on alginate/chitosan microparticles for BCG encapsulation

The aim of the present study was to develop novel Mycobacterium bovis bacille Calmette-Guérin (BCG)-loaded polymeric microparticles with optimized particle surface characteristics and biocompatibility, so that whole live attenuated bacteria could be further used for pre-exposure vaccination against Mycobacterium tuberculosis by the intranasal route. BCG was encapsulated in chitosan and alginate microparticles through three different polyionic complexation methods by high speed stirring. For comparison purposes, similar formulations were prepared with high shear homogenization and sonication. Additional optimization studies were conducted with polymers of different quality specifications in a wide range of pH values, and with three different cryoprotectors. Particle morphology, size distribution, encapsulation efficiency, surface charge, physicochemical properties and biocompatibility were assessed. Particles exhibited a micrometer size and a spherical morphology. Chitosan addition to BCG shifted the bacilli surface charge from negative zeta potential values to strongly positive ones. Chitosan of low molecular weight produced particle suspensions of lower size distribution and higher stability, allowing efficient BCG encapsulation and biocompatibility. Particle formulation consistency was improved when the availability of functional groups from alginate and chitosan was close to stoichiometric proportion. Thus, the herein described microparticulate system constitutes a promising strategy to deliver BCG vaccine by the intranasal route.

Microfluidic Encapsulation of Pickering Oil Microdroplets into Alginate Microgels for Lipophilic Compound Delivery

Alginate microgels are widely used as delivery systems in food, cosmetics, and pharmaceutical industries for encapsulation and sustained release of hydrophilic compounds and cells. However, the encapsulation of lipophilic molecules inside these microgels remains a great challenge because of the complex oil-core matrix required. The present study describes an original two-step approach allowing the easy encapsulation of several oil microdroplets within alginate microgels. In the first step, stable oil microdroplets were formed by preparing an oil-in-water (O/W) Pickering emulsion. To stabilize this emulsion, we used two solid particles, namely the cotton cellulose nanocrystals (CNC) and calcium carbonate (CaCO3). It was observed that the surface of the oil microdroplets formed was totally covered by a CNC layer, whereas CaCO3 particles were adsorbed onto the cellulose layer. This solid CNC shell efficiently stabilized the oil microdroplets, preventing them from undesired coalescence. In the second step, oil microdroplets resulting from the Pickering emulsion were encapsulated within alginate microgels using microfluidics. Precisely, the outermost layer of oil microdroplets composed of CaCO3 particles was used to initiate alginate gelation inside the microfluidic device, following the internal gelation mode. The released Ca2+ ions induced the gel formation through physical cross-linking with alginate molecules. This innovative and easy to carry out two-step approach was successfully developed to fabricate monodisperse alginate microgels of 85 pm in diameter containing around 12 oil microdroplets of 15 mu m in diameter. These new oil-core alginate microgels represent an attractive system for encapsulation of lipophilic compounds such as vitamins, aroma compounds or anticancer drugs that could be applied in various domains including food, cosmetics, and medical applications.

Micoencapsulation of bioactive plant extracts: a tool to functionalize cottage cheeses

Foeniculum vulgare Mill. (fennel) and Matricaria recutita L. (chamomile) are two examples of plants with reported antioxidant and antimicrobial properties, which can be related with their composition in phenolic compounds [1,2]. Furthermore, according to previous results of our research group, the direct incorporation of the aqueous extracts showed capacity to maintain the nutritional properties of the cottage cheeses, up to 7 days of storage, while improving the antioxidant potential. However, after 14 days, a decrease in the antioxidant properties was observed [1,2], which can be related with factors such as light, moisture, temperature and pH, that can cause bioactive compounds degradation. Therefore, the aim of the present study was to prepare microcapsules with the aqueous extracts of fennel and chamomile for incorporation in cottage cheese samples, in order to protect the bioactive molecules present in the extracts, such as phenolic compounds, and prevent the decrease of the antioxidant activity observed after the 14 days period. The microspheres were prepared using an atomization/coagulation technique. Sodium alginate was used as the matrix material to produce the microspheres that were characterized through optical microscopy (OM), during and after atomization, for inspecting morphology. The encapsulation efficiency (EE) was determined by HPLC-DAD by an indirect method by analysing the coagulation solution. FTIR was also used to attest the presence of the extract inside of the alginate matrix. These microencapsulated extracts were incorporated in cottage cheese samples that were further characterized in terms of nutritional properties and antioxidant potential right after incorporation, and after 7 and 14 days of storage at 4•c. The EE was estimated as -100% and the FTIR analysis confirmed the presence of the extracts inside the microspheres. The results showed that the incorporation of the microencapsulated extracts did not cause changes in the nutritional value of cottage cheeses (through a comparison with control samples without extracts). The predominant fatty acids were palmitic (C16:0) and oleic (CI8:0) acids. The order of abundance of fatty acids was as follows: saturated fatty acids (SF A)> monounsaturatcd fatty acids (MUF A)> polyunsaturated fatty acids (PUF A). Regarding free sugars, lactose was the only sugar identified and quantified in all samples. Regarding the antioxidant activity, the samples functionalized with the microencapsulated extracts showed a higher preservation of this property even after the 7th day of storage. Overall, the incorporation of the protected plant extracts in dairy foods can be a strategy to provide health benefits to consumers.

Polymeric alginate nanoparticles containing the local anesthetic bupivacaine

Bupivacaine (BVC; S75-R25, NovaBupialginate is a water-soluble linear polysaccharide. The present study reports the development of alginate/bis(2-ethylhexyl) sulfosuccinate (AOT) and alginate/chitosan nanoparticle formulations containing BVC (0.5%). The amounts of BVC associated in the alginate/AOT and alginate/chitosan nanoparticles were 87 +/- 1.5 and 76 +/- 0.9%, respectively. The average diameters and zeta potentials of the nanoparticles were measured for 30 days, and the results demonstrated the good stability of these particles in solution. The in vitro release kinetics showed a different behavior for the release profile of BVC in solution, compared with BVC-loaded alginate nanoparticles. In vitro and in vivo assays showed that alginate-chitosan BVC (BVC(ALG-CHIT)) and alginate-AOT BVC (BVC(ALG-AOT)) presented low cytotoxicity in 3T3-fibroblasts, enhanced the intensity, and prolonged the duration of motor and sensory blockades in a sciatic nerve blockade model.

Paraquat-loaded alginate/chitosan nanoparticles: Preparation, characterization and soil sorption studies

Agrochemicals are amongst the contaminants most widely encountered in surface and subterranean hydrological systems. They comprise a variety of molecules, with properties that confer differing degrees of persistence and mobility in the environment, as well as different toxic, carcinogenic, mutagenic and teratogenic potentials, which can affect non-target organisms including man. In this work, alginate/chitosan nanoparticles were prepared as a carrier system for the herbicide paraquat. The preparation and physicochemical characterization of the nanoparticles was followed by evaluation of zeta potential, pH, size and polydispersion. The techniques employed included transmission electron microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy. The formulation presented a size distribution of 635 +/- 12 nm, polydispersion of 0.518, zeta potential of -22.8 +/- 2.3 mV and association efficiency of 74.2%. There were significant differences between the release profiles of free paraquat and the herbicide associated with the alginate/chitosan nanoparticles. Tests showed that soil sorption of paraquat, either free or associated with the nanoparticles. was dependent on the quantity of organic matter present. The results presented in this work show that association of paraquat with alginate/chitosan nanoparticles alters the release profile of the herbicide, as well as its interaction with the soil, indicating that this system could be an effective means of reducing negative impacts caused by paraquat. (C) 2011 Elsevier B.V. All rights reserved.

On-demand generation and removal of alginate biocompatible microvalves for flow control in microfluidics

[EN] This paper describes, for the first time, the use of alginate hydrogels as miniaturised microvalves within microfluidic devices. These biocompatible and biodegradable microvalves are generated in situ and on demand, allowing for microfluidic flow control. The microfluidic devices were fabricated using an origami inspired technique of folding several layers of cyclic olefin polymer followed by thermocompression bonding. The hydrogels can be dehydrated at mild temperatures, 37◦C, to slightly open the microvalve and chemically erased using an ethylenediaminetetraacetic acid disodium salt (EDTA) solution, to completely open the channel, ensuring the reusability of the whole device and removal of damaged or defective valves for subsequent regeneration.

Survival and fermentation activity of probiotic bacteria and oxidative stability of omega-3 oil in co-microcapsules during storage

Tuna oil (O) and probiotic bacteria Lactobacillus casei (P) were co-microencapsulated in whey protein isolate (WPI)-gum Arabic (GA) complex coacervate. The co-microcapsules (WPI-P-O-GA), L. casei microcapsules (WPI-P-GA) and tuna oil microcapsules (WPI-O-GA) were converted into powder using spray and freeze drying. The interaction between probiotic bacteria and omega-3 oil in co-microcapsules, particularly in terms of oxidative stability of omega-3 oil and vitality/viability of probiotic bacteria and any synergistic outcome, was studied. The effect of storage temperature (5 and 25 °C) and time (90 days) on the survival and fermentation activity of L. casei and oxidative stability of tuna oil in the microcapsules/co-microcapsules was determined. A synergism between oxidative stability of omega-3 oil and vitality of probiotic bacteria was observed, when they were co-microencapsulated and spray dried. These co-microcapsules will likely have utility in functional food formulations due to simple and cost effective stabilisation and delivery of two important functional ingredients.

Kinetic analysis of metal uptake by dry and gel alginate particles

The kinetics of metal uptake by gel and dry calcium alginate beads was analysed using solutions of copper or lead ions. Gel beads sorbed metal ions faster than the dry ones and larger diffusivities of metal ions were calculated for gel beads: approximately 10−4 cm2/min vs. 10−6 cm2/min for dry beads. In accordance, scanning electron microscopy and nitrogen adsorption data revealed a low porosity of dry alginate particles. However, dry beads showed higher sorption capacities and a mechanical stability more suitable for large-scale use. Two sorption models were fitted to the kinetic results: the Lagergren pseudo-first order and the Ho and McKay pseudo-second order equations. The former was found to be the most adequate to model metal uptake by dry alginate beads and kinetic constants in the orders of 10−3 and 10−2 min−1 were obtained for lead solutions with concentrations up to 100 g/m3. The pseudo-first order model was also found to be valid to describe biosorbent operation with a real wastewater indicating that it can be used to design processes of metal sorption with alginate-based materials.

Evaluation of dry protonated calcium alginate beads for biosorption applications and studies of lead uptake

Alginate polysaccharide is a promising biosorbent for metal uptake. Dry protonated calcium alginate beads for biosorption applications were prepared, briefly characterized and tested for lead uptake. Several advantages of this biosorbent are reported and discussed in comparison with other alginate-based sorbents. The alginate beads contained 4.7 mmol/g of COOH groups, which suffered hydrolysis near pH 4. The Weber and Morris model, applied to kinetic results of lead uptake, showed that intraparticle diffusion was the rate-controlling step in lead sorption by dry alginate beads. Equilibrium experiments were performed and the data were fitted with different isotherm models. The Langmuir equation was the most adequate to model lead sorption. The maximum uptake capacity (qmax) was estimated as 339 mg/g and the Langmuir constant (b) as 0.84 l/mg. These values were compared with that of other sorbents found in the literature, indicating that dry protonated calcium alginate beads are among the best biosorbents for the treatment and recovery of heavy metals from aqueous streams.

Evaluation of dry protonated calcium alginate beads for biosorption applications and studies of lead uptake

Alginate polysaccharide is a promising biosorbent for metal uptake. Dry protonated calcium alginate beads for biosorption applications were prepared, briefly characterized and tested for lead uptake. Several advantages of this biosorbent are reported and discussed in comparison with other alginate-based sorbents. The alginate beads contained 4.7 mmol/g of COOH groups, which suffered hydrolysis near pH 4. The Weber and Morris model, applied to kinetic results of lead uptake, showed that intraparticle diffusion was the rate-controlling step in lead sorption by dry alginate beads. Equilibrium experiments were performed and the data were fitted with different isotherm models. The Langmuir equation was the most adequate to model lead sorption. The maximum uptake capacity (qmax) was estimated as 339 mg/g and the Langmuir constant (b) as 0.84 l/mg. These values were compared with that of other sorbents found in the literature, indicating that dry protonated calcium alginate beads are among the best biosorbents for the treatment and recovery of heavy metals from aqueous streams.

Copper ions binding in cu‐alginate gelation

Alginate polysaccharide forms viscous aqueous dispersions and has the ability to form gels in the presence of divalent cations such as calcium and copper. In this work, we have studied cooper ions binding during Cu‐alginate gelation, obtaining quantitative information about the amount and kinetics of cation binding. Our results indicate that copper binding during gelation occurs until a Langmuir‐type equilibrium is reached between bound and free ions in the gel‐contacting solution. The kinetics of metal ions binding can be modeled using Ritchie equation–derived models, allowing the prediction of ionic binding and gel formation temporal evolution. The ratio between cationic and polysaccharide quantities in the gelation system determines the kinetics of gelation and the characteristics of the gel formed. The experimental results and models applied in the work give more insights on alginate gelation and contribute to a reliable design and control of production methods for alginate gel structures.