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In this chapter, we review different types of nanocarrier systems, their architecture, how and to what extent they are capable to penetrate the skin to deliver different bioactive molecules.

Diversos parásitos y patógenos afectan la productividad y la supervivencia de las abejas melíferas. Los metabolitos secundarios de las plantas son tratamientos alternativos potenciales, sin embargo, su efecto ha sido poco estudiado sobre las enfermedades microsporidianas. Además, hay poca información sobre la toxicidad resultante de la administración oral prolongada de estas sustancias a las abejas. En esta investigación, evaluamos los efectos in vivo de diferentes aceites esenciales y componentes principales (CPs) sobre la supervivencia de abejas y el desarrollo de Nosema ceranae bajo un régimen de ad libitum sin elección. Las sustancias administradas en el jarabe de sacarosa a las abejas recién emergidas a diferentes concentraciones se consumieron con avidez y causaron diferentes resultados de supervivencia. No obstante, las dosis subletales de sustancias no controlaron el parásito. Diverse parasites and pathogens affect productivity and survival of honey bees. Plant secondary metabolites are poten- tial alternative treatments, however, their effect has been little studied on microsporidian diseases. Furthermore, there is poor information about the toxicity resulting from prolonged oral administration of these substances to bees. In this research, we evaluated in vivo effects of different essential oils and main components (MCs) on bee survival and Nosema ceranae development under an ad libitum non-choice regimen. Substances administered on sucrose syrup to newly emerged bees at different concentrations were avidly consumed and caused different survival performances. Neverthe- less, sublethal doses of substances did not control the parasite. Fil: Porrini, Martín Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Garrido, Paula Melisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Gende, Liesel Brenda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Rossini, Cármen. Universidad de la República; Uruguay Fil: Hermida, Laura Graciela. Instituto Nacional de Tecnología Industrial; Argentina Fil: Marcangeli, Jorge Augusto. Universidad Nacional de Mar del Plata; Argentina Fil: Eguaras, Martin Javier. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; Argentina

Insect repellent textiles are currently being investigated as a complementary tool to control disease vectors, such as mosquitoes. The application of microencapsulated biopesticides to textiles is a promising low-toxicity alternative to other control methods, as microcapsules may prolong the release of the repellent agent. In this work, microcapsules containing two biopesticides, namely citronella essential oil and citriodiol® were prepared and applied to cotton textiles using a variety of techniques. The aim of this work was to evaluate these functional textiles to select the most efficient, durable systems. Citriodiol-treated cotton presented a prolonged durability, and 100% repellency could be achieved for more than 30 days after padding onto cotton fabrics. It has to be pointed out that all these repellent textiles have been obtained by scalable, low-cost methods which would require no additional investment for textile finishing industries., Facultad de Ciencias Naturales y Museo, Centro de Estudios Parasitológicos y de Vectores

In the present study, two methods were used to evaluate the in vitro release of leuprolide acetate (LA) from poly(lac tide-co-glycolide) (PLGA) microspheres: Franz diffusion cells, typically referred to as “vertical diffusion cells” (VDC), and rotating bottle apparatus (RBA), both modified with a dialysis membrane. This hydrosoluble peptide was chosen as a model drug to study different possibilities of in vitro testing and analyze the variables that affect drug release, respecting sink and physiological conditions. Microspheres were prepared with a conventional double emulsion–solvent evaporation method using PLGA (50:50) with a relatively low molecular weight. Comprehensive stability tests for LA were performed in the conditions used for in vitro release assays. In phosphate-buffered saline (PBS), LA showed no significant degradation, but in an acidic medium, it degraded dramatically. The release profile of the delivery system was governed mainly by diffusion as explained by the low molecular weight of the polymer and the high water solubility of the peptide. The in vitro release profiles were triphasic in vertical diffusion cells and biphasic in the rotating bottle apparatus. The release kinetics was enhanced in RBA with respect to VDC, probably because the constant movement of a suspension of loose microspheres in a large volume and the large membrane area facilitated drug migration. The smoother, triphasic profiles obtained with VDC can be explained by the partial confinement of microspheres, which is similar to the described in vivo behavior of an injectable delivery system.

Functional textiles are being developed in order to provide fabrics with new properties and added value. They can be obtained either by using new chemical fibers or by incorporating functional agents to conventional fabrics. Microencapsulation is an effective method to protect these functional agents from reactions with moisture, light, and oxygen. If a fabric is treated with microencapsulated functional agents, higher durability of functionality is expected. This article reports the development and testing of two types of microcapsules containing essential oils for application in cotton fabrics. Microcapsules were obtained by complex coacervation using gelatin and arabic gum or by encapsulation in yeast cells in order to increase the durability of fragrances in textiles. Microcapsule characterization, such as particle size and morphology, was carried out for different oils to polymer ratios and hardening agents to polymer ratios. Padding and coating were tested as application methods. The morphology, durability of the fragrance, and laundering properties of the treated fabrics were investigated. The use of an electronic nose to measure the fragrance release from microcapsules was also evaluated. Gelatin—arabic gum microcapsules increased the durability of the fragrance on the treated fabrics and withstood one wash cycle. Fabrics treated with yeast cell microcapsules presented low fragrance intensity before washing. The fragrance was not detectable after laundering, even though the microcapsules could still be observed on the fabric.

ALA administration has been used to induce the endogenous photosensitiser Protoporphyrin IX for photodynamic therapy (PDT) of tumours. However, the hydrophilic nature of ALA limits its ability to penetrate through skin restricting the use of ALA-PDT to superficial diseases. Lipophilic derivatives of ALA such as ALA Undecanoyl ester (Und-ALA) were designed to have better diffusing properties. However, Und-ALA, applied topically on the skin over the tumour, induced low porphyrin content. To improve Und-ALA efficacy we tested the efficacy of Und-ALA as porphyrin inducer, delivered in phosphatidylcholine and phosphatidylglycerol (PC-PG) or phosphatidylcholine and phosphatidic acid (PC-PA) liposomal formulations. Entrapment of Und-ALA into PC-PA or PC-PG liposomes resulted in a dramatic impairment of toxicity in the mammary tumour LM3 cells. However, liposomal Und-ALA induced lower intracellular porphyrin content compared to free ALA, although total porphyrins content (intracellular+media) from free Und-ALA resulted equal compared to liposomal Und-ALA, due to induction of porphyrins release induced by the latter. Topical administration of Und-ALA in PC-PG or PC-PA liposomes over the skin of LM3 subcutaneously injected mice, induced equal amount of tumour porphyrins as compared to free Und-ALA. The kinetics of porphyrins synthesis from Und-ALA is similar for free and liposomal formulations both in vivo and in vitro, showing that release of Und-ALA from liposomes is not gradual and suggesting that liposome membranes either fuses or binds to the cell membranes. To sum up, the incorporation of Und-ALA into liposomes of PC-PA or PC-PG composition does not improve the rate of porphyrin synthesis either in vitro or in vivo, due to a massive release of extracellular porphyrins and a poor cytoplasmatic release of the liposome content. The design of new liposome compositions either favouring endocytosis or coated with natural polymers to prevent Und-ALA interaction with cellular membrane are desired to overcome intracellular porphyrin release after long-chained ALA esters treatment.

Three types of pyranine (HPTS)-containing liposomes were prepared by high-pressure homogenization under optimized conditions. At 37 degrees C, they were 1) fluid-state vesicles made from soybean phosphatidylcholine (SPC), 2) gel-state liposomes made from hydrogenated SPC (HSPC), and 3) solid-disordered membranes obtained from HSPC and cholesterol (HSPC-Chol). These liposome formulations were characterized before, during, and after in vitro digestion, which involved the presence of pH gradients, enzymes, and bile salts. Mean sizes and size distributions of the vesicles were determined by DLS; (31)P-NMR (nuclear magnetic resonance) was used to quantify lyso-PC forms; internal pH was monitored throughout digestion with two different fluorescent pH probes; and changes in bilayer permeability and HPTS encapsulation were determined by size-exclusion chromatography and fluorimetry. Differential scanning calorimetry analysis was also performed in order to study the effect of digestion on HSPC vesicles. SPC liposomes were physically stable during digestion; they presented 8% lyso-forms and an HPTS encapsulation around 85% after in vitro digestion. However, they were extremely permeable to ions, so that the internal pH immediately equilibrated with the bulk pH. HSPC liposomes were the most affected by the digestive process. Even though they were chemically stable, as inferred from the low lyso-PC content, very important changes in their size distribution were observed. A final 50% HPTS leakage was quantified after in vitro digestion. Nevertheless, they were the least permeable to protons under pH gradients. HSPC-Chol vesicles presented intermediate permeability to protons, having their internal pH decreased from approximately 6.8 to 4.6 after 1 hour of incubation at pH 2. This was the most chemically stable formulation and showed the highest encapsulation, even after in vitro digestion. Therefore, HSPC-Chol liposomes would be the most adequate choice for the design of lipid products for oral administration.

Health benefits associated with omega-3 (ω-3) fatty acid consumption have been extensively demonstrated. Moreover, the absence of ω-3 fatty acids in a normal diet, particularly those known as long chain polyunsaturated fatty acids (PUFAs), has been related to a wide variety of diseases. Even though they can be easily obtained from animal and vegetable sources, typical Western diets contain low levels of long chain ω-3 fatty acids. This has led to the development of different kinds of PUFA-fortified foods to increase the uptake of these essential lipids. Microencapsulation has been introduced as an effective strategy to incorporate polyunsaturated ω-3 oils into foods and to avoid their oxidation during manufacture and storage. Many technologies have been developed for oil encapsulation, some of which have been successfully scaled up. Microencapsulated oil powders are now commercialized, and ω-3 containing functional foods are starting to spread worldwide.

The use of liposomes for oral administration of drugs and for food applications is based on their ability to preserve entrapped substances and to increase their bioavailability. Bile salts are one of the agents that affect the liposome structure during intestinal digestion and the main reported studies on liposome/bile salt systems used only one bile salt. The aim of this work is to characterise the interaction of liposomes with a natural bile salt extract (BSE) at physiological pH and temperature. Three types of liposomes (fluid, gel-state and liquid-ordered bilayers) were studied. Phase diagrams were obtained and a very different behaviour was found. Fluid bilayers were completely permeable to an entrapped dye with partial or complete disruption of vesicles (final size 10 nm). Gel-state bilayers released their content but BSE led to the formation of large mixed structures (2000 nm). Liquid-ordered bilayers formed mixed vesicles (1000 nm) and, surprisingly, retained a high percentage of their aqueous content (about 50%). As a consequence, each type of liposome offers singular features to be used in oral applications due to their specific interaction with bile salts.

Health benefits associated to ω-3 fatty acids consumption together with the high susceptibility to oxidation of ω-3 containing oils have led to the development of microencapsulated oils for nutraceutical and food enrichment applications. The aim of this work is to obtain different formulations for linseed oil microencapsulation by spray-drying with high encapsulation efficiency and evaluate their resistance to oxidation through the accelerated Rancimat test. Four formulations were tested; using different combinations of gum arabic (GA), maltodextrin (MD), methyl cellulose (MC) and whey protein isolate (WPI). Microcapsules made of 100% GA and ternary mixtures of GA, MD and WPI presented the highest protection from oxidation and microencapsulation efficiencies higher than 90%. They also presented spherical structures with smooth surfaces which kept unaltered after 10-month storage. GA containing formulation was included in bread manufacturing. Fortified bread resulted similar in appearance to control bread without microcapsules, but α-linolenic acid content was reduced significantly after preparation. Fil: Gallardo, Gabriela. Instituto Nacional de Tecnología Industrial; Argentina Fil: Guida, Leticia. Instituto Nacional de Tecnología Industrial; Argentina Fil: Martinez, Vanina. Instituto Nacional de Tecnología Industrial; Argentina Fil: López, Maria C.. Instituto Nacional de Tecnología Industrial; Argentina Fil: Bernhardt, Dana Cecilia. Instituto Nacional de Tecnología Industrial; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Blasco, Ramiro. Instituto Nacional de Tecnología Industrial; Argentina Fil: Pedroza Islas, Ruth. Universidad Iberoamericana de México; México Fil: Hermida, Laura Graciela. Instituto Nacional de Tecnología Industrial; Argentina

The aim of this work was to study the iron uptake of Caco-2 cells incubated with five different formulations of liposomes containing iron. The vesicles were also characterized before, during, and after in vitro digestion. Caco-2 cells were incubated with digested and nondigested liposomes, and soluble iron uptake was determined. Nondigested liposomes made with chitosan (CHI) or the cationic lipid, DC-Cholesterol (DC-CHOL), generated the highest iron uptake. However, these two formulations were highly unstable under in vitro digestion, resulting in nonmeasurable iron uptake. Digested conventional liposomes composed of soybean phosphatidylcholine (SPC), hydrogentated phosphatidylcholine (HSPC), or HSPC and cholesterol (CHOL) presented the highest iron-uptake values. These liposomal formulations protected iron from oxidation and improved iron uptake from intestinal cells, compared to an aqueous solution of ferrous sulphate.

Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease of cattle caused by Moraxella bovis (Mb). Parenterally administered immunogens used to prevent the disease do not offer complete protection possibly because they stimulate a poor ocular mucosal secretory response, in which locally secreted immunoglobulin-A (sIgA) is one of the main components. The principal aim of this study was to evaluate by an indirect enzyme linked immunosorbent assay (ELISA), the local ocular mucosal sIgA response against Mb purified pili, produced after intranasal inoculation of experimental vaccines. Pili were adjuvanted by several different adjuvants (QuilA, Marcol Arlacel, Marcol Span, microencapsulated pili with PLGA polymers). Results were compared to sIgA response produced by adjuvant placebo inoculations and by IBK natural infection. Significantly higher anti-pili IgA response (p0.05) was detected in calves vaccinated intranasally with pili QuilA and pili Marcol Span compared to control calves, although this specific immune response did not seem to be related to protection against Mb infection or typical IBK lesion development.

Microcapsules containing citronella essential oil were prepared by complex coacervation and applied to cotton textiles in order to study the repellent efficacy of the obtained fabrics. Citronella released from treated textiles was indirectly monitored by the extractable content of its main components. Repellent activity was assessed by exposure of a human hand and arm covered with the treated textiles to Aedes aegypti mosquitoes. Fabrics treated with microencapsulated citronella presented a higher and longer lasting protection from insects compared to fabrics sprayed with an ethanol solution of the essential oil, assuring a repellent effect higher than 90% for three weeks. Complex coacervation is a simple, low cost, scalable and reproducible method of obtaining encapsulated essential oils for textile application. Repellent textiles were achieved by padding cotton fabrics with microcapsules slurries using a conventional pad-dry method. This methodology requires no additional investment for textile finishing industries, which is a desirable factor in developing countries.

Liposomes of different compositions have been designed to improve delivery of aminolevulinic acid (ALA) and its esterified derivatives ALA-Hexyl ester (He-ALA) and ALA-Undecanoyl ester (Und-ALA) for its use in photodynamic therapy (PDT). Egg yolk phosphatidyl choline (PC), phosphatidic acid (PA) and phosphatidyl glycerol (PG) were employed in the preparation of the liposomes. Sonicated vesicles composed of PC, PC–PG (80:20) or PC–PA (80:20) containing ALA or derivatives were obtained and purified by a minicolumn centrifugation method. PC liposomes presented encapsulation percentages around 6% for 2 mM ALA, 13% for 2 mM He-ALA and 51% for 2 mM Und-ALA. The addition of PG or PA to the formulation, resulted in an increased entrapment: 19% for 2 mM ALA, 69% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC–PG liposomes and 21% for 2 mM ALA, 60% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC–PA liposomes. Higher concentrations of ALA or derivatives resulted in lower percentages of entrapment. The three formulations containing ALA or derivatives were stable up to 1 week upon storage at 4 °C. However, upon dilution with medium, ALA leaked from the liposomes, while on the contrary, He-ALA was highly retained, being therefore a good choice for its use in PDT. The stability of Und-ALA upon dilution could not be tested, but Und-ALA proved to have the highest entrapment efficacy.

Superparamagnetic iron oxide nanoparticles coated with natural polymers have found many applications in the field of biosensors as magnetic carriers due to the combination of two characteristics: the possibility of controlling the particles movement by applying external magnetic fields and attaching biomolecules to the particle through a chemical bond. The aim of this work was to prepare long-term stabilized particles with acid groups available to be used as magnetic carriers of biomolecules. In this study, we present the synthesis of maghemite nanoparticles coated with alginate, a natural polymer. Particles were characterized by electron microscopy. Magnetic properties were studied by vibrating sample magnetometry, which revealed the superparamagnetic behavior of maghemite nanoparticles. Horseradish peroxidase (HRP) was chemically bonded to the maghemite-alginate particles and the enzymatic activity of HRP was determined by a colorimetric technique. The maghemite-alginate particles were immobilized onto the surface of a gold electrode by means of a magnet and the HRP activity was followed electrochemically, showing that these particles can be successfully used in amperometric sensors. Furthermore, maghemite-alginate particles were also used for the diagnosis of the foot-and-mouth disease by means of an enzyme-linked immunoassay with electrochemical detection. 3ABC protein, a non-structural protein of the virus, was linked to the maghemite-alginate particles and used to selectively detect anti-3ABC antibodies in cattle sera.