Green diesel synthesis by hydrodeoxygenation of lipids extracted from Chlorella algae in supercritical hexane

Lipids were extracted from Chlorella algae with supercritical hexane. The high lipids yield of approximately 10% was obtained at optimum conditions of 300 rpm stirring speed and 2 h duration compared to the total contents of lipids being 12%. Furthermore, an easiness of hexane recovery may be considered as economically and ecologically attractive. For the first time, in the current work catalytic hydrodeoxygenation (HDO) of Chlorella algal lipids was studied over 5 wt% Ni/H-Y-80 and 5 wt% Ni/SiO2 at 300 C and under 30 bar total pressure in H2. A comparative HDO of stearic acid was carried out under similar conditions. The conversion of lipids was about 35% over 5 wt% Ni/H-Y-80 after 6h, whereas, 5 wt% Ni/SiO2 was totally deactivated after 60 min. The selectivity to hydrocarbons (C15-C18) is 6%. As a comparison, complete conversion of stearic acid over 5 wt% Ni/H-Y-80 was achieved in 6 h. The transformation of lipids proceeded mostly via hydrogenation and hydrolysis with formation of free fatty acid (FFA). The lower activity might be attributed to deactivation of catalysts caused by chlorophylls and carotenoids. Even though the conversion is low, future studies in HDO of lipids extracted from other algae species having higher lipid content could be proposed. Coke resistant catalyst might be considered to improve catalytic activity.

Development And Characterization Of A Cationic Lipid Nanocarrier As Non-viral Vector For Gene Therapy.

The aim of the present work was to produce a cationic solid lipid nanoparticle (SLN) as non-viral vector for protein delivery. Cationic SLN were produced by double emulsion method, composed of softisan(®) 100, cetyltrimethylammonium bromide (CTAB), Tween(®) 80, Span(®) 80, glycerol and lipoid(®) S75 loading insulin as model protein. The formulation was characterized in terms of mean hydrodynamic diameter (z-ave), polydispersity index (PI), zeta potential (ZP), stability during storage time, stability after lyophilization, effect of toxicity and transfection ability in HeLa cells, in vitro release profile and morphology. SLN were stable for 30days and showed minimal changes in their physicochemical properties after lyophilization. The particles exhibited a relatively slow release, spherical morphology and were able to transfect HeLa cells, but toxicity remained an obstacle. Results suggest that SLN are nevertheless promising for delivery of proteins or nucleic acids for gene therapy.

Comparative physical-chemical characterization of encapsulated lipid-based isotretinoin products assessed by particle size distribution and thermal behavior analyses

Isotretinoin is the drug of choice for the management of severe recalcitrant nodular acne. Nevertheless, some of its physical-chemical properties are still poorly known. Hence, the aim of our study consisted to comparatively evaluate the particle size distribution (PSD) and characterize the thermal behavior of the three encapsulated isotretinoin products in oil suspension (one reference and two generics) commercialized in Brazil. Here, we show that the PSD, estimated by laser diffraction and by polarized light microscopy, differed between the generics and the reference product. However, the thermal behavior of the three products, determined by thermogravimetry (TGA), differential thermal (DTA) analyses and differential scanning calorimetry (DSC), displayed no significant changes and were more thermostable than the isotretinoin standard used as internal control. Thus, our study suggests that PSD analyses in isotretinoin lipid-based formulations should be routinely performed in order to improve their quality and bioavailability. (C) 2010 Elsevier B.V. All rights reserved.

Commercial squids: Characterization, assessment of potential health benefits/risks and discrimination based on mineral, lipid and vitamin E concentrations

The most consumed squid species worldwide were characterized regarding their concentrations of minerals, fatty acids, cholesterol and vitamin E. Interspecific comparisons were assessed among species and geographical origin. The health benefits derived from squid consumption were assessed based on daily minerals intake and on nutritional lipid quality indexes. Squids contribute significantly to daily intake of several macro (Na, K, Mg and P) and micronutrients (Cu, Zn and Ni). Despite their low fat concentration, they are rich in long-chain omega-3 fatty acids, particularly docosahexaenoic (DHA) and eicosapentanoic (EPA) acids, with highly favorable ω-3/ω-6 ratios (from 5.7 to 17.7), reducing the significance of their high cholesterol concentration (140–549 mg/100 g ww). Assessment of potential health risks based on minerals intake, non-carcinogenic and carcinogenic risks indicated that Loligo gahi (from Atlantic Ocean), Loligo opalescens (from Pacific Ocean) and Loligo duvaucelii (from Indic Ocean) should be eaten with moderation due to the high concentrations of Cu and/or Cd. Canonical discriminant analysis identified the major fatty acids (C14:0, C18:0, C18:1, C18:3ω-3, C20:4ω-6 and C22:5ω-6), P, K, Cu and vitamin E as chemical discriminators for the selected species. These elements and compounds exhibited the potential to prove authenticity of the commercially relevant squid species.

Development and characterization of lipid-based nanosystems for controlled release of bioactive compounds

Tese de Doutoramento em Engenharia Química e Biológica.

Characterization of an interaction between fetal hemoglobin and lipid A of LPS resulting in augmented induction of cytokine production in vivo and in vitro.

A previously described extract of sheep fetal liver was reported to reverse many of the cytokine changes associated with aging in mice, including an augmented spleen cell ConA-stimulated production of IL-4 and decreased production of IL-2. Similar effects were not seen with adult liver preparations. These changes were observed in various strains of mice, including BALB/c, DBA/2 and C57BL/6, using mice with ages ranging from 8 to 110 weeks. Preliminary characterization of this crude extract showed evidence for the presence of Hb gamma chain, as well as of lipid A of LPS. We show below that purified preparations of sheep fetal Hb, but not adult Hb, in concert with suboptimally stimulating doses of LPS (lipid A), cooperate in the regulation of production of a number of cytokines, including TNFalpha and IL-6, in vitro. Furthermore, isolated fresh spleen or peritoneal cells from animals treated in vivo with the same combination of Hb and LPS, showed an augmented capacity to produce these cytokines on further culture in vitro. Evidence was also obtained for a further interaction between CLP, LPS and fetal Hb itself in this augmented cytokine production. These data suggest that some of the functional activities in the fetal liver extract reported earlier can be explained in terms of a novel immunomodulatory role of a mixture of LPS (lipid A) and fetal Hb.

Characterization of N-cadherin association to lipid rafts in melanoma

RESUME La peau est un organe complex composé de deux parties distinctes: l'épiderme et le derme, séparé par une membrane basale. Dans la couche basale de l'épiderme, les melanocytes synthétisent la mélanine dans des mélanosomes. Les mélanosomes sont ensuite transportés des mélanocytes vers les kératinocytes, protégeant ainsi la peau des dégâts dus aux radiations U.V. La E-cadhérine assure l'adhésion entre les mélanocytes et les kératinocytes. Au cours de la transformation du mélanocyte en cellule malignes, les mélanocytes perdent l'expression de la E-cadhérine et, simultanément, se mettent à exprimer la N-cadhérine, ce phénomène est nommé « cadherin switch ». La perte de l'expression de la E-cadhérine permet au mélanocytes d'échapper au contrôle des kératinocytes, tandis que l'expression de la N-cadhérine promeut l'invasion métastasique des cellules de mélanome. Préalablement, nous avons trouvé qu'une fraction de la N-cadhérine était localisée les microdomaines membranaires spécialisés, enrichi en cholestérol et en glycosphingolipides, appelés « lipid rafts ». Une des particularité des « lipid rafts » est qu'ils sont riches en molécules permettant la transmission de signaux d'activation. De plus, des travaux récents rapportent qu'un sous-type de « lipid rafts » appelé caveolae pourrai contribuer à la progression tumorale. S'appuyant sur le rôle prépondérant de la N-cadhérine dans la progression du mélanome ainsi que sur sa présence dans les « lipid rafts », nous avons émis l'hypothèse que l'association de la N-cadhérine avec les « lipid rafts » pourrai contribuer à la progression du mélanome. Le but de ce projet à été de caractériser l'association de la Ncadhérine avec les « lipid rafts » au cours de la progression du mélanome. Au moyen de lignées cellulaires humaines, dérivées de mélanomes à différents stades de progression, nous avons trouvé que (1) la N-cadhérine est partiellement associée aux «lipid rafts » dans six lignées dérivées de mélanome en phase avancée de progression et dans des tumeurs expérimentales, mais pas dans deux lignées dérivées de mélanome à un stade plus précoce ; (2) l'association de la N-cadhérine dans les « lipid rafts » ne dépent pas de son niveau d'expression ; (3) la E-cadhérine n'est pas présente dans les « lipid rafts »d'une lignée de cellule de mélanome ayant conservé l'expression de la E-cadhérine ; (4) la localisation de la N-cadhérine dans les « lipid rafts »n'est pas modulée par les facteurs de croissance bFGF, IGF-I, et HRG1-β1, ni par des voies de signalisation impliquant MEK, PKA, les kinases de la famille Src, et PI3K ; (5) l'association de la N-cadhérine avec les « lipid rafts » n'est pas requise pour la stabilisation des jonctions adhérentes et n'est pas perturbée par la destruction de ces dernières ; (6) la N-cadhérine dans les « lipid rafts » forme un complexe avec β-caténine, p 120ctn et α-caténine. En conclusion, cette étude originale montre pour la première fois que dans des cellules de mélanome agressifs, une fraction de la N-cadhérine est localisée dans les « lipid rafts » en association avec β-caténine, p 120ctn et α-caténine. Comme la présence de la N-cadhérine dans les « lipid rafts » ne contribue pas à la formation de jonction adhérentes, cette étude suggère une nouvelle fonction pour la N-cadhérine dans les « lipid rafts ». SUMMARY Human skin is a complex organ composed of two layers separated by a basement membrane: the epidermis and the dermis. In the basal layer of the epidermis, the melanin-producing cells of the skin, the melanocytes deliver melanin-containing melanosomes to keratinocytes, thereby protecting the epidermis and the dermis from the deleterious effects of ultraviolet light. Melanocytes physically interact with keratinocytes through E-cadherin-mediated adhesion. During malignant transformation into melanoma cells, melanocytes lose E-cadherin expression and concomitantly gain expression of N-cadherin, a phenomenon referred to as "cadherin switch". Loss of E-cadherin allows melanocytes to escape the regulatory effects of neighbouring keratinocytes, while gain of N-cadherin expression promotes migration, invasion and metastatic abilities of melanoma cells. In preliminary experiments, we found that a fraction of N-cadherin localized to specialized membrane microdomains enriched in cholesterol- and glycosphingolipid, called lipid rafts. One particular feature of lipid rafts is that they are rich in signalling molecules and they possibly modulate transmembrane signalling events. Moreover, recent reports suggested that a specialized type of rafts called caveolae might contribute to tumor progression. Based on the documented role of N-cadherin in melanoma progression and its presence in lipid rafts of melanoma cells, we raised the hypothesis that the association of N-cadherin with lipid rafts might be relevant to melanoma progression. The aim of this project was to characterize N-cadherin associated to lipid rafts during melanoma progression. Using human melanoma cell lines derived from melanoma at different stages of progression, we found that (1) N-cadherin is partly associated to lipid rafts in six cell lines derived from melanomas at late stages of progression and in experimental tumors, but not in two melanoma cell lines derived from early stages; (2) N-cadherin targeting to lipid rafts does not depend on its expression level; (3) E-cadherin is not localized in lipid rafts of a melanoma cell line that retained E-cadherin expression; (4) N-cadherin localization to lipid rafts is not modulated by the growth factors bFGF, IGF-I, and HRG1-β1, nor by MEK-, PKA-, Src family kinases-, and PI3K-mediated signalling events; (5) the association of N-cadherin with lipid rafts is not required for adherens junctions stability nor it is perturbed by adherens junctions disruption; (6) N-cadherin in lipid rafts is in complex with β-catenin, p 120ctm and α-catenin. In conclusion, this study provides original evidence that in aggressive melanoma cells a pool of N-cadherin is localized in lipid rafts in association with β-catenin, p 120 and α-catenin. The presence of N-cadherin in lipid rafts independently of its involvement in adherens junctions formation, suggests a possible new role for N-cadherin recruited to lipid rafts. Further studies investigating the biological meaning of this localization promise to uncover new properties of this molecule.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56% lipids and 9 and 7% carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

Production and characterization of lipid microparticles produced by spray cooling encapsulating a low molar mass hydrophilic compound

The objective of this research was to produce and characterize lipid particles (MpLs) that may be used as carriers of high amounts of hydrophilic core and evaluate the influence of the core amount on the performance of lipid microparticles. The MpLs were produced by spray cooling from solid and liquid lipid mixtures (stearic and oleic fatty acids and partly hydrogenated vegetable fat) containing glucose solution as core and soy lecithin as surfactant. The performance of MpLs was evaluated by means of the effective amount of encapsulated core, the core amount present on the surface of MpLs (superficial glucose) and the core release profile in aqueous solution. Morphological observations showed that MpLs presented spherical shape and a rugged and continuous surface, and an average diameter between 25 and 32 µm. The effective amount of encapsulated core was greater than 78% for all formulations evaluated. Larger amounts of superficial glucose were found in formulations in which more concentrated glucose solutions were used, regardless of the glucose lipid-solution ratio. The release results showed that core retention was significantly influenced by the glucose solution concentration, whereas release modulation was influenced by the glucose lipid-solution ratio.

Characterization, physicochemical stability, and evaluation of in vitro digestibility of solid lipid microparticles produced with palm kernel oil and tristearin

Solid lipid particles have been investigated by food researchers due to their ability to enhance the incorporation and bioavailability of lipophilic bioactives in aqueous formulations. The objectives of this study were to evaluate the physicochemical stability and digestibility of lipid microparticles produced with tristearin and palm kernel oil. The motivation for conducting this study was the fact that mixing lipids can prevent the expulsion of the bioactive from the lipid core and enhance the digestibility of lipid structures. The lipid microparticles containing different palm kernel oil contents were stable after 60 days of storage according to the particle size and zeta potential data. Their calorimetric behavior indicated that they were composed of a very heterogeneous lipid matrix. Lipid microparticles were stable under various conditions of ionic strength, sugar concentration, temperature, and pH. Digestibility assays indicated no differences in the release of free fatty acids, which was approximately 30% in all analises. The in vitro digestibility tests showed that the amount of palm kernel in the particles did not affect the percentage of lipolysis, probably due to the high amount of surfactants used and/or the solid state of the microparticles.

NMR characterization of chlorhexidine in lipid-based formulations /

A mixture of Chlorhexidine digluconate (CHG) with glycerophospholipid 1,2-dimyristoyl- <^54-glycero-3-phospocholine (DMPC-rf54) was analysed using ^H nuclear magnetic resonance. To analyze powder spectra, the de-Pake-ing technique was used. The method is able to extract simultaneously both the orientation distribution function and the anisotropy distribution function. The spectral moments, average order parameter profiles, and longitudinal and transverse relaxation times were used to explore the structural phase behaviour of various DMPC/CHG mixtures in the temperature range 5-60°C.

Characterization of Lipid Droplets and Functional Analysis of Lipid Droplet-Associated Proteins in Dictyostelium discoideum

Lipid droplets (LDs) are the universal storage form of fat as a reservoir of metabolic energy in animals, plants, bacteria and single celled eukaryotes. Dictyostelium LD formation was investigated in response to the addition of different nutrients to the growth medium. LDs were induced by adding exogenous cholesterol, palmitic acid (PA) as well as growth in bacterial suspension, while glucose addition fails to form LDs. Among these nutrients, PA addition is most effective to stimulate LD formation, and depletion of PA from the medium caused LD degradation. The neutral lipids incorporated into the LD-core are composed of triacylglycerol (TAG), steryl esters, and an unknown neutral lipid (UKL) species when the cells were loaded simultaneously with cholesterol and PA. In order to avoid the contamination with other cellular organelles, the LD-purification method was modified. The isolated LD fraction was analysed by mass spectrometry and 100 proteins were identified. Nineteen of these appear to be directly involved in lipid metabolism or function in regulating LD morphology. Together with a previous study, a total of 13 proteins from the LD-proteome were confirmed to localize to LDs after the induction with PA. Among the identified LD-proteins, the localization of Ldp (lipid droplet membrane protein), GPAT3 (glycerol-3-phosphate acyltransferase 3) and AGPAT3 (1-acylglycerol-3-phosphate-acyltransferase 3) were further verified by GFP-tagging at the N-termini or C-termini of the respective proteins. Fluorescence microscopy demonstrated that PA-treatment stimulated the translocation of the three proteins from the ER to LDs. In order to clarify DGAT (diacylglycerol acyltransferase) function in Dictyostelium, the localization of DGAT1, that is not present in LD-proteome, was also investigated. GFP-tagged DGAT1 localized to the ER both, in the presence and absence of PA, which is different from the previously observed localization of GFP-tagged DGAT2, which almost exclusively binds to LDs. The investigation of the cellular neutral lipid level helps to elucidate the mechanism responsible for LD-formation in Dictyostelium cells. Ldp and two short-chain dehydrogenases, ADH (alcohol dehydrogenase) and Ali (ADH-like protein), are not involved in neutral lipid biosynthesis. GPAT, AGPAT and DGAT are three transferases responsible for the three acylation steps of de novo TAG synthesis. Knock-out (KO) of AGPAT3 and DGAT2 did not affect storage-fat formation significantly, whereas cells lacking GPAT3 or DGAT1 decreased TAG and LD accumulation dramatically. Furthermore, DGAT1 is responsible for the accumulation of the unknown lipid UKL. Overexpression of DGAT2 can rescue the reduced TAG content of the DGAT1-KO mutant, but fails to restore UKL content in these cells, indicating that of DGAT1 and DGAT2 have overlapping functions in TAG synthesis, but the role in UKL formation is unique to DGAT1. Both GPAT3 and DGAT1 affect phagocytic activity. Mutation of GPAT3 increases it but a DGAT1-KO decreases phagocytosis. The double knockout of DGAT1 and 2 also impairs the ability to grow on a bacterial lawn, which again can be rescued by overexpression of DGAT2. These and other results are incorporated into a new model, which proposes that up-regulation of phagocytosis serves to replenish precursor molecules of membrane lipid synthesis, whereas phagocytosis is down-regulated when excess fatty acids are used for storage-fat formation.  

Novel immunoadjuvants based on cationic lipid: Preparation, characterization and activity in vivo

The interactions between three different protein antigens and dioctadecyldimethylammonium bromide (DODAB) dispersed in aqueous solutions from probe sonication or adsorbed its one bilayer onto particles was comparatively investigated. The three model proteins were bovine serum albumin (BSA), purified 18 kDa/14 kDa antigens from Taenia crassiceps (18/14-Tcra) and a recombinant, heat-shock protein hsp-18 kDa from Mycobacterium leprae. Protein-DODAB complexes in water solution were characterized by dynamic light scattering for sizing and zeta-potential analysis. Cationic complexes (80-100 nm of mean hydrodynamic diameter) displayed sizes similar to those of DODAB bilayer fragments (BF) in aqueous solution and good colloid stability over a range of DODAB and protein concentrations. The amount of cationic lipid required for attaining zero of zeta-potential at a given protein amount depended on protein nature being smaller for 18 kDa/14 kDa antigens than for BSA. Mean diameters for DODAB/protein complexes increased, whereas zeta-potentials decreased with NaCl or protein concentration. In mice, weak IgG production but significant cellular immune responses were induced by the complexes in comparison to antigens alone or carried by aluminum hydroxide as shown from IgG in serum determined by ELISA, delayed type hypersensitivity reaction from footpad swelling tests and cytokines analysis. The novel cationic adjuvant/protein complexes revealed good colloid stability and potential for vaccine design at a reduced DODAB concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Thermal characterization of solid lipid nanoparticles containing praziquantel

Solid lipid nanoparticles (SLNs), loaded and unloaded with praziquantel (PRZ-load SLN and PRZ-unload SLN) were prepared by two different procedures: (a) oil-in-water hot microemulsion method, obtaining at 70 degrees C an optically transparent blend composed of surfactant, co-surfactant, and water; and (b) oil-in-water microemulsion method, dissolving the lipid in an immiscible organic solvent, emulsified in water containing surfactants and co-surfactant, and then evaporated under reduced pressure at 50 degrees C. The mean diameter, polydispersity index (PdI), and zeta potential were 187 to 665 nm, 0.300 to 0.655, and -25 to -28 mV respectively, depending on the preparation method. The components, binary mixture, SLNs loaded and unloaded with PRZ, and physical mixture were evaluated by differential scanning calorimetry (DSC) and thermogravimetry (TG). The non-isothermal isoconversional Flynn-Wall-Ozawa method was used to determine the kinetic parameters associated with the thermal decomposition of the samples. The experimental data indicated a linear relationship between the apparent activation energy E and the pre-exponential factor A, also called the kinetic compensation effect (KCE), allowing us to determine the stability with respect to the preparation method. Loading with PRZ increased the thermal stability of the SLNs.