One-pot synthesis of alginate-antimicrobial peptide nanogel.

Nanosized alginate-based particles (NAPs) were obtained in a one-pot solvent-free synthesis procedure, achieving the design of a biocompatible nanocarrier for the encapsulation of IbM6 antimicrobial peptide (IbM6). IbM6 is integrated in the nascent nanosized hydrogel self-assembly guided by electrostatic interactions and by weak interactions, typical of soft matter. The formation of the nanogel is a dynamic and complex process, which presents an interesting temporal evolution. In this work, we optimized the synthesis conditions of IbM6-NAPs based on small-angle X-ray scattering (SAXS) measurements and evaluated its time evolution over several weeks by sensing the IbM6 environment in IbM6-NAPs from photochemical experiments. Fluorescence deactivation experiments revealed that the accessibility of different quenchers to the IbM6 peptide embedded in NAPs is dependent on the aging time of the alginate network. Lifetimes measurements indicate that the deactivation paths of the excited state of the IbM6 in the nanoaggregates are reduced when compared with those exhibited by the peptide in aqueous solution, and are also dependent on the aging time of the nanosized alginate network. Finally, the entrapment of IbM6 in NAPs hinders the degradation of the peptide by trypsin, increasing its antimicrobial activity against Escherichia coli K-12 in simulated operation conditions.

Bacteria metabolic adaptation to oxidative stress: the case of silica.

Although the presence of silica in many living organisms offers advanced properties including cell protection, the different in vitro attempts to build living materials in pure silica never favoured the cells viability. Thus, little attention has been paid to host-guest interactions to modify the expected biologic response. Here we report the physiological changes undergone by Escherichia coli K-12 in silica from colloidal solution to gel confinement. We show that the physiological alterations in growing cultures are not triggered by the initial oxidative Reactive Oxygen Species (ROS) response. Silica promotes the induction of alternative metabolic pathways along with an increase of growth suggesting the existence of rpoS polymorphisms. Since the functionality of hybrid materials depends on the specific biologic responses of their guests, such cell physiological adaptation opens perspectives in the design of bioactive devices attracting for a large field of sciences.

Development of a biosensor for environmental monitoring based on microalgae immobilized in silica hydrogels.

A new biosensor was designed for the assessment of aquatic environment quality. Three microalgae were used as toxicity bioindicators: Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii. These microalgae were immobilized in alginate and silica hydrogels in a two step procedure. After studying the growth rate of entrapped cells, chlorophyll fluorescence was measured after exposure to (3-(3,4-dichlorophenyl)-1,1-dimethylurea) (DCMU) and various concentrations of the common herbicide atrazine. Microalgae are very sensitive to herbicides and detection of fluorescence enhancement with very good efficiency was realized. The best detection limit was 0.1 µM, obtained with the strain C. reinhardtii after 40 minutes of exposure.

Design of silica nanocarriers: Tuning the release to embryonic stem cells by simple strategies.

The design of mesoporous silica nanoparticles (MSNs) for drug delivery is attracting increasing interest. Controlled release of their cargo is usually mediated by diffusion and erosion mechanisms, which might not reach the expected therapeutic effects. Here, we report the development and characterization of MSNs which modulate the cargo release in different cell models: fibroblasts and embryonic stem cells. Based on a double strategy: the presence of multimodal pore channels and a chitosan coating, we demonstrated a modulated release. Our results show that MSNs could be used for controlled drug delivery in different cell types, showing the potential of improving pluripotent stem cells differentiation and reprogramming protocols with promising applications in biomedicine.

Bioaccessibility assay, antioxidant activity and consumer-oriented sensory analysis of Beta vulgaris by-product encapsulated in Ca(II)-alginate beads for different foods.

Bioaccessibility analysis and antioxidant activity along in vitro digestion and a consumer-oriented sensory analysis were conducted in three potential functional foods based on Ca(II)-alginate beads containing bioactive compounds extracted from beet stems. Ca(II)-alginate beads per se, and two selected products (cookies and turkish delights supplemented with the beads) were prepared. Regarding the beads, among the attributes rated by consumers, visual appreciation predominates, being color in the just-as-right (JAR) category and in the like preference. Instead, both flavor and sweet taste were attributes highly penalized and should be improved in beads to be accepted as food per se. A higher percentage of customers preferred cookies and turkish delights instead of only beads, considering global satisfaction. Regarding in vitro digestion, there was a significant content of phenolic compounds in the products with beads, showing a bioaccessibility greater than 80% (for cookies) and 26% (for turkish delights). Also, the antioxidant capacity measured by ABTS ranged between 50 and 109% for cookies and turkish delights, being lower when measured by FRAP (between 20 and 30%, respectively). Thus, including the beads with beet stem extract in both products leads to a significant increase in the content of phenolic compounds and in the antioxidant capacity compared to their counterparts, protecting the compound during oral and gastric phases. These results allow the generation of improved Ca(II)-alginate systems with promising functional properties for the development of ingredients and functional foods.

Evaluation of calcium alginate bead formation kinetics: An integrated analysis through light microscopy, rheology and microstructural SAXS.

Ca(II)-alginate beads are being produced for a broad spectrum of biotechnological uses. Despite the simplicity of their manufacturing process, in these highly complex arrangements, the final properties of the material strongly depend on the supramolecular scaffolding. Here we present a cost-effective automatized Optical Video Microscopy approach for in situ evaluation of the kinetics of alginate bead formation. With simple mathematic modeling of the acquired data, we obtained key parameters that reveal valuable information on the system: the time course of gel-front migration correlates with the plateau of the storage module, and total volume shrinkage is highly related to the stabilization of shear strain and shear stress at the yield point. Our results provide feasible and reproducible tools, which allow for a better interpretation of bead formation kinetics and a rapid screening technique to use while designing gelling materials with specific properties for technological applications.

Effects of in vitro digestion-fermentation over global antioxidant response and short chain fatty acid production of beet waste extracts in Ca(ii)-alginate beads.

The aim of the present work was to analyze the effect of in vitro gastrointestinal digestion-fermentation on antioxidant capacity, total phenols and production of short chain fatty acids (SCFAs) from biocompounds derived from beet waste (leaf and stem) encapsulated in different formulations of Ca(ii)-alginate beads. The encapsulated systems presented higher antioxidant capacity in different phases (digested and fermented) than the extracts without encapsulation, making Ca(ii)-alginate beads a suitable delivery vehicle. Levels of total phenolic compounds and antioxidant capacity of the fermented fraction were up to ten times higher than those of the digested fraction, boosted by the contribution of bioactive compounds from the by-product of beet as well as by sugars and biopolymers. Among the formulations used, those that had excipients (sugars and/or biopolymers) presented a better overall antioxidant response than the beads with just alginate. Guar gum and sucrose lead to a promising enhancement of Ca(ii)-alginate beads not only for preservation and protection but also in terms of stability under in vitro digestion-fermentation and production of SCFAs.

Effect of in vitro digestion-fermentation of Ca(II)-alginate beads containing sugar and biopolymers over global antioxidant response and short chain fatty acids production.

The aim of the present paper was to unravel the effect of a standardized in vitro European protocol of digestion-fermentation over Ca(II)-alginate beads synthesized with sugars and biopolymers. Special emphasis on the antioxidant capacity using methods that simulate physiological conditions, short-chain fatty acids (SCFAs) production, and a detailed study of the microstructure of the gel network by SAXS at several scales (1-100 nm) were considered. Beads released high antioxidant capacity during digestion assessed by several methods, comparable to some common foods; antioxidant capacity was improved with sucrose and arabic gum inclusion in the formulation. After fermentation by gut microbiota, a ten-fold increase in the antioxidant values and an important SCFAs production were obtained, revealing the enhanced ability to produce these functional biomolecules. The microstructural analysis of Ca(II)-alginate showed an advantageous behavior: they slightly changed in oral and gastric fluids and partially dissolved their structure in intestinal fluid, where absorption occurs.

Biopolymeric pellets of polyvinyl alcohol and alginate for the encapsulation of Ib-M6 peptide and its antimicrobial activity against E. coli.

The encapsulation of Ib-M6 antibacterial peptide in pellets of polyvinyl alcohol (PVA) and polyvinyl alcohol-alginate (PVA-Alg) matrices was carried out in order to explore its controlled release and activity against Escherichia coli K-12. The pellets were obtained by combined ice segregation induced self-assembly (ISISA) and freezing-thawing methods and their microstructure was studied by scanning electron microscopy. Bromothymol blue was used as a model compound to study the transport mechanisms and release from pellets. The results show that there is a significant effect of the total concentration of PVA precursor solutions, the mass ratio of PVA of different molecular weights and the addition of alginate on the microstructure and transport properties of pellets. The antibacterial activity of Ib-M6 against Escherichia coli K-12 was not affected by the encapsulation in PVA pellets. However, the release of Ib-M6 from PVA-Alg pellets was not possible, probably due to the electrostatic interaction of positively charged Ib-M6 and negatively alginate structure. Nonetheless, the controlled release of Ib-M6 from polymeric matrices can be fitting by modifying parameters such as the concentration and type of polymer precursors.

Effects of pH, extrusion tip size and storage protocol on the structural properties of Ca(II)-alginate beads.

The Ca(II)-alginate beads were formulated changing some synthesis variables: pH (3.8-6.8), extrusion tip size (0.25-0.50 mm) and washing/storage protocol, to evaluate possible implications in the structural properties of beads, which are critical to scale and standardize their production at industrial level. Regardless of the macro- (diameter, area, perimeter and roundness, studied by image analysis) and micro-structural (size, density and interconnectivity of rods assessed by SAXS) parameters analyzed, there are no effects related to the washing and storage protocol employed for any synthesis conditions. Structural parameters are only influenced by the synthesis pH. Both washing protocol and extrusion tip size effects on the consolidation of the alginate network are negligible at each pH value. Besides, none of the synthesis variables affected the availability of water within the beads as assessed by diffusion coefficient and water activity measurements. A model, relating chain-chain interactions and polymer chain packing, is proposed.

Gums induced microstructure stability in Ca(II)-alginate beads containing lactase analyzed by SAXS.

Previous works show that the addition of trehalose and gums in ß-galactosidase (lactase) Ca(II)-alginate encapsulation systems improved its intrinsic stability against freezing and dehydration processes in the pristine state. However, there is no available information on the evolution in microstructure due to the constraints imposed by the operational conditions. The aim of this research is to study the time course of microstructural changes of Ca(II)-alginate matrices driven by the presence of trehalose, arabic and guar gums as excipients and to discuss how these changes influence the diffusional transport (assessed by LF-NMR) and the enzymatic activity of the encapsulated lactase. The structural modifications at different scales were assessed by SAXS. The incorporation of gums as second excipients induces a significant stabilization in the microstructure not only at the rod scale, but also in the characteristic size and density of alginate dimers (basic units of construction of rods) and the degree of interconnection of rods at a larger scale, improving the performance in terms of lactase activity.

Plant cell proliferation inside an inorganic host.

In recent years, much attention has been paid to plant cell culture as a tool for the production of secondary metabolites and the expression of recombinant proteins. Plant cell immobilization offers many advantages for biotechnological processes. However, the most extended matrices employed, such as calcium-alginate, cannot fully protect entrapped cells. Sol-gel chemistry of silicates has emerged as an outstanding strategy to obtain biomaterials in which living cells are truly protected. This field of research is rapidly developing and a large number of bacteria and yeast-entrapping ceramics have already been designed for different applications. But even mild thermal and chemical conditions employed in sol-gel synthesis may result harmful to cells of higher organisms. Here we present a method for the immobilization of plant cells that allows cell growth at cavities created inside a silica matrix. Plant cell proliferation was monitored for a 6-month period, at the end of which plant calli of more than 1 mm in diameter were observed inside the inorganic host. The resulting hybrid device had good mechanical stability and proved to be an effective barrier against biological contamination, suggesting that it could be employed for long-term plant cell entrapment applications.

Ca(ii) and Ce(iii) homogeneous alginate hydrogels from the parent alginic acid precursor: a structural study.

Alginate hydrogels are suitable for the encapsulation of biomolecules and microorganisms for the building of bioactive materials. Several alternatives to the conventional alginate formulation are being studied for a broad range of biotechnological applications; among them the crosslinking of alginate by lanthanide cations, Ln(iii), envisages expanded biomedical applications. The performance of these functional materials is highly related to the microstructure of the alginate matrix, which in turn is affected by the conditions of synthesis. In particular, when a diffusing gradient of the crosslinking cation is involved, microstructure inhomogeneities are expected at the macroscopic level. Here we discuss the subtle differences in the microstructure, as assessed by SAXS (Small Angle X-ray Scattering), established in the direction of the gradient of diffusion of Ca(ii) or Ce(iii).

Feasibility of using a translucid inorganic hydrogel to build a biosensor using immobilized algal cells.

Anthropic activities generate contaminants, as pesticides and other pollutants, in the aquatic environment which present a real threat to ecosystems and human health. Thus, monitoring tools become essential for water managers to detect these chemicals before the occurrence of adverse effects. In this aim, algal cell biosensors, based on photosystem II activity measurement, have been designed for several years in previous studies. In this work, we study a new immobilization technique of algal cells in the aim of improving the performance of these biosensors. Immobilization was here achieved by encapsulation in a hybrid alginate/silica translucid hydrogel. The feasibility of this process was here assessed, and the biosensor designed was tested on the detection of chemicals in urban rainwaters.

Silica@proton-alginate microreactors: a versatile platform for cell encapsulation.

As an alternative approach to the well known Ca(ii)-alginate encapsulation process within silica hydrogels, proton-driven alginate gelation was investigated in order to establish its capacity as a culture carrier, both isolated and embedded in an inorganic matrix. Control over the velocity of the proton-gelation front allows the formation of a hydrogel shell while the core remains liquid, allowing bacteria and microalgae to survive the strongly acidic encapsulation process. Once inside the inorganic host, synthesized by a sol-gel process, the capsules spontaneously redissolve without the aid of external complexing agents. The entrapped cells survive the two-step process to a significant extent; culture's growth restores the initial cell count in less than two weeks. Biosynthesis of Au nanoparticles mediated by the entrapped microalgae illustrates the preservation of the biosynthetic abilities supported by this platform.

Sol-gel silica platforms for microalgae-based optical biosensors.

An advanced hybrid biosensing platform with improved optical quality is developed based on the acidic encapsulation of microalgi in silica matrices synthesized by TAFR (tetraethoxysilane derived alcohol free route). The three microalgi (Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii) were previously immobilized in alginate following the two-step procedure. Tuning the alginate protecting function with the aid of Tris-HCl buffer, the sol-gel synthesis was conducted at pH 4.0 well below the tolerance limit imposed by the encapsulated microalgae. The acidic condensation of Si(IV) generates silica matrices with outstanding optical properties that suit the requirements of biosensors based on optical detection methods.

Rhodamine B doped silica encapsulation matrices for the protection of photosynthetic organisms.

An advanced encapsulation matrix that efficiently protects microalgae from harmful UV light without causing toxicity to the entrapped culture is developed based on the electrostatic adsorption of the dye Rhodamine B on silica preformed particles during sol-gel synthesis. The three microalgae (Chlorella vulgaris, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii) were previously immobilized in alginate following the Two-step procedure. Once entrapped in the silica gel, Rhodamine B act as an inner cut-off filter, protecting the encapsulated organisms from UV radiation. This matrix allows the sterilization of encapsulation devices without affecting the viability of the entrapped microalgae cells. The condensation of Si(IV) in the presence of silica particles with adsorbed dye generates silica matrices with good mechanical stability. Furthermore; no appreciable differences in microstructure, as assessed by SAXS (Small Angle X-ray Scattering), are caused by the addition of the dye.

Co-encapsulation of Daphnia magna and microalgae in silica matrices, a stepping stone toward a portable microcosm.

We report on the first silica encapsulation of a metazoan (Daphnia magna), with a high initial viability (96% of the population remained active 48 h after encapsulation). Moreover, the co-encapsulation of this crustacean and microalgae (Pseudokirchneriella subcapitata) was achieved, creating inside a silica monolith, the smallest microcosm developed to present. This artificial ecosystem in a greatly diminished scale isolated inside a silica nanoporous matrix could have applications in environmental monitoring, allowing ecotoxicity studies to be carried out in portable devices for on-line and in situ pollution level assessment.

New method for the simultaneous determination of diffusion and adsorption of dyes in silica hydrogels.

The fine tuning of porosity in sol gel based devices makes possible the design of novel applications in which the transport of molecules through the oxide gel plays a crucial role. In this work we develop a new method for the simultaneous analysis of diffusion and adsorption of small diffusing probes, as anionic and cationic dyes, through silica mesoporous hydrogels synthesized by sol-gel. The novelty of the work resides in the simplicity of acquisition of the experimental data (by means of a desk scanner) and further mathematical modeling, which is in line with high throughput screening procedures, enabling rapid and simultaneous determination of relevant diffusion and adsorption parameters. Net mass transport and adsorption properties of the silica based hydrogels were contrasted to dye adsorption isotherms and textural characterization of the wet gels by SAXS, as well as that of the corresponding aerogels determined by Field Emission Scanning Electron Microscopy (FESEM) and N2 adsorption. Thus, the validation of the results with well-established characterization methods demonstrates that our approach is robust enough to give reliable physicochemical information on these systems.