RESUMEN

Mature honeys that brew naturally in the hive develop distinct bioactive components, and thus carry a higher premium due to their superior quality. However, how to identify mature honeys remains difficult. Trace oligosaccharides are a likely source of biomarkers to indicate maturity. Here, we profiled trace oligosaccharides in acacia honey by GC-MS and used a metabolomics strategy to screen oligosaccharides that distinguish honeys with different maturities. Turanose content increased gradually in acacia honey samples and was closely related to the days stored in the hive (p < 0.05). To accurately quantify turanose, a UPLC-ELSD method was developed. Using the established method, honeys with ≥1.20 g/100 g of turanose could be classified as mature acacia honey. Based on the preliminary study, 500 commercial acacia honeys were analyzed, and only 77.2 % of these samples had a satisfactory level of turanose. This work offers a potential method to evaluate the quality of honeys.

Asunto(s)

Acacia , Miel , Cromatografía de Gases y Espectrometría de Masas , Miel/análisis , Metabolómica , Oligosacáridos

RESUMEN

In order to know the catalytic activities of the disaccharidases expressed in the mammalian small intestinal brush-border membrane vesicles (BBMV) high concentrated solutions of sucrose, maltose, isomaltulose, trehalose and the mixture sucrose:lactose were incubated with pig small intestine disaccharidases. The hydrolysis and transglycosylation reactions generated new di- and trisaccharides, characterized and quantified by GC-MS and NMR, except for trehalose where only hydrolysis was detected. In general, α-glucosyl-glucoses and α-glucosyl-fructoses were the most abundant structures, whereas no fructosyl-fructoses or fructosyl-glucoses were found. The in-depth structural characterization of the obtained carbohydrates represents a new alternative to understand the potential catalytic activities of pig small intestinal disaccharidases. The hypothesis that the oligosaccharides synthesized by glycoside hydrolases could be also hydrolysed by the same enzymes was confirmed. This information could be extremely useful in the design of new non-digestible or partially digestible oligosaccharides with potential prebiotic properties.

Asunto(s)

Glicósido Hidrolasas , Intestino Delgado , Animales , Hidrólisis , Microvellosidades , Oligosacáridos , Porcinos

RESUMEN

Fifty potato genotypes from twenty-four different countries of origin, four different flesh colours (yellow, purple, red and marble) and different cultivation types (Andean accessions, landraces, breeder lines and cultivated varieties) were studied in terms of their nutritional and physicochemical characteristics. In general, cultivated varieties and breeder lines showed the highest similarity (slight differences only in some particular fatty acids distributions: C10:0, C12:0 and C22:0) concerning the physicochemical parameters assayed in this work, independently of the geographical origin or tuber flesh colour of these genotypes. Nonetheless, some of the studied landraces and Andean accessions proved to be similar enough to be considered as genotypes with good potential for commercial cultivation. These results can contribute to the supply of new potato genotypes into sustainable farming systems, supporting the protection of potato biodiversity, particularly Andean accessions, landraces and coloured genotypes (red or purple flesh) which are not widely cultivated so far.

Asunto(s)

Biodiversidad , Fenómenos Químicos , Genotipo , Valor Nutritivo , Solanum tuberosum/química , Análisis Discriminante , Tubérculos de la Planta/química , Solanum tuberosum/clasificación

RESUMEN

Stress granules (SGs) are assemblies of mRNA and proteins that form from mRNAs stalled in translation initiation in response to stress. Chronic stress might even induce formation of cytotoxic pathological SGs. SGs participate in various biological functions including response to apoptosis, inflammation, immune modulation, and signalling pathways; moreover, SGs are involved in pathogenesis of neurodegenerative diseases, viral infection, aging, cancers and many other diseases. Emerging evidence has shown that small molecules can affect SG dynamics, including assembly, disassembly, maintenance and clearance. Thus, targeting SGs is a potential therapeutic strategy for the treatment of human diseases and the promotion of health. The established methods for detecting SGs provided ready tools for large-scale screening of agents that alter the dynamics of SGs. Here, we describe the effects of small molecules on SG assembly, disassembly, and their roles in the disease. Moreover, we provide perspective for the possible application of small molecules targeting SGs in the treatment of human diseases.

Asunto(s)

Antineoplásicos/farmacología , Antivirales/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Proteínas/metabolismo , ARN Mensajero/metabolismo , Estrés Fisiológico , Virosis/tratamiento farmacológico , Envejecimiento , Animales , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/patología , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , ARN Mensajero/genética , Transducción de Señal , Virosis/genética , Virosis/metabolismo , Virosis/patología

RESUMEN

Lactose is mostly hydrolysed in the small intestine, whereas lactulose, recognised prebiotic carbohydrate, reaches the colon to be fermented by the intestinal microbiota. Digestibility of these substrates was investigated by an in vitro digestion model using a Rat Small Intestine Extract (RSIE). A kinetic study of lactose digestion showed levels of hydrolysis (82.8%) at 0.2 mg*mL-1 and the highest hydrolysis rate constant (kobt). Considering these conditions, lactulose showed high resistance to intestinal digestion by RSIE, resulting in low hydrolysis degrees (20.4%) after 5 h reaction. These results underline the suitability of these intestinal extracts under the studied conditions, as a reliable tool to evaluate carbohydrate digestion and support the evidences towards the higher resistance of galactosyl-fructose linkages during its intestinal degradation.

Asunto(s)

Glicósido Hidrolasas/metabolismo , Intestino Delgado/metabolismo , Lactosa/metabolismo , Lactulosa/metabolismo , Animales , Microbioma Gastrointestinal , Hidrólisis , Cinética , Ratas

RESUMEN

Cryoprotective saccharides are widely accepted antifreeze additives that reduce thawing loss, maintain texture, and retard protein denaturation in frozen seafood. In this study, the inhibition effects of trehalose and alginate oligosaccharides on ice growth were investigated and compared with sodium pyrophosphate (Na4P2O7) treatment in peeled shrimp during frozen storage, especially the interactions between saccharide molecules and ice crystals. The microstructural results demonstrated that the pre-soaking of trehalose and alginate oligosaccharides before freezing exhibited marked effects on stability of muscle tissue structures and slowed the damage caused to the myofibrils by large ice crystals. The ice-growth inhibition activities might play an important role in cryoprotective effects of saccharides on frozen muscle tissue. Furthermore, molecular docking and molecular dynamics (MD) simulations proved that saccharides were generally close to the ice interface and embedded in ice layers via hydrogen bonds or hydrophobic or electrostatic interactions. The saccharides-ice complex was partially destroyed, and some dislocation and disaggregation were observed around the saccharides molecules. Thus, the incorporated saccharides suppressed the growth of ice crystals, providing protection from freeze-induced damage. Here, the obtained structural details of the ice crystals interface affected by trehalose and alginate oligosaccharides were well in agreement with the histological (H&E staining) experimental results. These findings help better understand the ice-growth inhibition mechanisms of saccharides in frozen shrimp, and these two saccharides may be potentially used as ice-growth inhibitors in frozen seafood.

Asunto(s)

Congelación , Hielo/análisis , Oligosacáridos/química , Trehalosa/química , Alginatos/química , Animales , Difosfatos/química , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Penaeidae/química , Electricidad Estática

RESUMEN

Lactobacillus buchneri is a Gram-positive, obligate heterofermentative, facultative anaerobe commonly affiliated with spoilage of food products. Notably, L. buchneri is able to metabolize lactic acid into acetic acid and 1,2-propanediol. Although beneficial to the silage industry, this metabolic capability is detrimental to preservation of cucumbers by fermentation. The objective of this study was to characterize isolates of L. buchneri purified from both industrial and experimental fermented cucumber after the onset of secondary fermentation. Genotypic and phenotypic characterization included 16S rRNA sequencing, DiversiLab® rep-PCR, colony morphology, API 50 CH carbohydrate analysis, and ability to degrade lactic acid in modified MRS and fermented cucumber media. Distinct groups of isolates were identified with differing colony morphologies that varied in color (translucent white to opaque yellow), diameter (1 mm-11 mm), and shape (umbonate, flat, circular or irregular). Growth rates in MRS revealed strain differences, and a wide spectrum of carbon source utilization was observed. Some strains were able to ferment as many as 21 of 49 tested carbon sources, including inulin, fucose, gentiobiose, lactose, mannitol, potassium ketogluconate, saccharose, raffinose, galactose, and xylose, while others metabolized as few as eight carbohydrates as the sole source of carbon. All isolates degraded lactic acid in both fermented cucumber medium and modified MRS, but exhibited differences in the rate and extent of lactate degradation. Isolates clustered into eight distinct groups based on rep-PCR fingerprints with 20 of 36 of the isolates exhibiting >97% similarity. Although isolated from similar environmental niches, significant phenotypic and genotypic diversity was found among the L. buchneri cultures. A collection of unique L. buchneri strains was identified and characterized, providing the basis for further analysis of metabolic and genomic capabilities of this species to enable control of lactic acid degradation in fermented plant materials.

Asunto(s)

Ácido Acético/metabolismo , Cucumis sativus/microbiología , Ácido Láctico/metabolismo , Lactobacillus/genética , Lactobacillus/metabolismo , Propilenglicol/metabolismo , Reactores Biológicos , Fermentación , Genotipo , Lactobacillus/clasificación , Lactobacillus/aislamiento & purificación , ARN Ribosómico 16S/genética

RESUMEN

The effects of hydrocolloids (hydroxypropylmethylcellulose (HPMC), Carboxymethylcellulose (CMC), xanthan gum (XG), and apple pectin (AP)) at different concentrations on dough thermo-mechanical properties and in vitro starch digestibility of gluten-free potato steamed bread were investigated. Results showed that hydrocolloids addition significantly increased the gelatinization temperature (from 52.0 to 64.2°C) and water absorption (from 56.22 to 66.50%) of potato dough. Moreover, hydrocolloids may be interacted with protein and starch, the density of potato protein bands was decreased by hydrocolloids addition, the reason might be that higher molecular weight complexes might be formed between proteins-hydrocolloids or proteins-proteins, thus change the protein solubility. Furthermore, steamed breads with hydrocolloids presented higher specific volume and lower hardness, and the rapidly digestible starch and estimated glycemic index were significantly decreased from 45.51 to 20.64, from 69.54 to 55.17, respectively. In conclusion, HPMC and XG could be used as improvers in the gluten-free potato steamed bread.

Asunto(s)

Solanum tuberosum , Almidón/química , Pan , Coloides , Harina , Glútenes

RESUMEN

pH shift-induced aggregation is frequently observed in downstream processing of monoclonal antibodies and has been shown to depend on solvent composition. To quantify the stabilizing effect of polyol additives against aggregation, we determined aggregation rate constants in the presence of a set of 14 compounds. Rate constants were then correlated with molecular descriptors in a quantitative structure activity relationship (QSAR) approach. The molecular size, volume, the charge, number of hydrogen acceptors, the stereochemistry and hydrophobicity of the compounds were identified as important descriptors. Generally larger compounds with a balanced surface polarity tend to inhibit aggregation better while hydrophobicity plays an important role at the nucleation phase, with hydrophobic compounds being more potent at inhibiting aggregation.

Asunto(s)

Anticuerpos Monoclonales/química , Polímeros/química , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Relación Estructura-Actividad Cuantitativa , Solventes

RESUMEN

Many lead compounds have a low solubility in water, which substantially hinders their clinical application. Nanosuspensions have been considered a promising strategy for the delivery of water-insoluble drugs. Here, denatured soy protein isolate (SPI)-coated docetaxel nanosuspensions (DTX-NS) were developed using an anti-solvent precipitation-ultrasonication method to improve the water-solubility of DTX, thus improving its intracellular delivery. DTX-NS, with a diameter of 150-250nm and drug-loading up to 18.18%, were successfully prepared by coating drug particles with SPI. Interestingly, the drug state of DTX-NS was alterable. Amorphous drug nanoparticles were obtained at low drug-loading, whereas at a high drug-loading, the DTX-NS drug was mainly present in the crystalline state. Moreover, DTX-NS could be internalized at high levels by cancer cells and enter the cytosol by lysosomal escape, enhancing cell cytotoxicity and apoptosis compared with free DTX. Taken together, denatured SPI has a strong stabilization effect on nanosuspensions, and the drug state in SPI-coated nanosuspensions is alterable by changing the drug-loading. Moreover, DTX-NS could achieve cytosolic delivery, generating enhanced cell cytotoxicity against cancer cells.

Asunto(s)

Antineoplásicos , Citosol/metabolismo , Portadores de Fármacos , Nanopartículas , Proteínas de Soja , Taxoides , Células A549 , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Docetaxel , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Liberación de Fármacos , Estabilidad de Medicamentos , Humanos , Interferón-alfa/sangre , Interleucina-12/sangre , Ratones Endogámicos ICR , Nanopartículas/administración & dosificación , Nanopartículas/química , Desnaturalización Proteica , Suero/química , Proteínas de Soja/administración & dosificación , Proteínas de Soja/química , Proteínas de Soja/farmacología , Taxoides/administración & dosificación , Taxoides/química , Taxoides/farmacología

RESUMEN

Blastose, a natural disaccharide found in honey, is usually found as a byproduct of fructo-oligosaccharide synthesis from sucrose with fructosyltransferases. In this study, we describe a novel two-step biosynthetic route to obtain blastose, designed from a detailed observation of B. subtilis levansucrase (SacB) acceptor structural requirements for fructosylation. The strategy consisted first in the synthesis of the trisaccharide O-ß-d-Fruf-(2↔6)-O-α-d-Glcp-(1↔1)-α-d-Glcp, through a regioselective ß-d-transfructosylation of trehalose (Tre) which acts as acceptor in a reaction catalyzed by SacB using sucrose or levan as fructosyl donor. In this reaction, levansucrase (LS) transfers regioselectively a fructosyl residue to either C6-OH group of the glucose residues in Tre. The resulting trisaccharide obtained in 23% molar yield based on trehalose, was purified and fully characterized by extensive NMR studies. In the second step, the trisaccharide is specifically hydrolyzed by trehalase, to obtain blastose in 43.2% molar yield based on the trisaccharide. This is the first report describing the formation of blastose through a sequential transfuctosylation-hydrolysis reaction.

Asunto(s)

Disacaridasas/metabolismo , Hexosiltransferasas/metabolismo , Trehalosa/metabolismo , Trisacáridos/metabolismo , Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Fructanos/metabolismo , Hidrólisis , Sacarosa/metabolismo

RESUMEN

Cryoprotective saccharides are widely accepted additives that reduce thawing loss, maintain texture, and retard protein denaturation in the frozen seafood. The present study aimed to investigate the roles of trehalose and alginate oligosaccharides on cryoprotection of frozen shrimp, primarily focusing on the interactions between myosin and saccharide molecules using a molecular dynamics (MD) simulation analysis. The results indicated that soaking in the trehalose and alginate oligosaccharides solutions markedly reduced thawing and cooking losses in frozen shrimp, with respective values decreasing to 6.02%, 8.14%, and 5.99%, 8.19% after 9weeks of storage, which were significantly lower than that of fresh water treatment (9.75% and 15.09%). Our assumption was that water replacement played a leading role in cryoprotection, as shown in previous experimental results and reports. Furthermore, homology modeling and MD simulations confirmed that trehalose and alginate oligosaccharides substituted the water molecules around the myosin surface by forming hydrogen bonds with polar residues of amino acids, thereby stabilizing the structures in the absence of water during frozen storage. These conditions affected the flexibility of particular amino acid residues, enhanced the residue cross correlations within the two chains of myosin, and also increased the total interaction energy between myosin and water/saccharide molecules, thereby leading to an increase in protein stability. Finally, by comparing the experimental results to that of MD simulation, significant positive correlation existed between saccharides and the stabilization of myosin in shrimp muscle. The findings of the present study may help better understand the cryoprotective mechanisms of saccharides in frozen shrimp, and the two saccharides may be potentially used as alternative additives in seafood to maintain better quality during frozen storage.

Asunto(s)

Alginatos/química , Oligosacáridos/química , Penaeidae/química , Alimentos Marinos/análisis , Trehalosa/química , Animales , Crioprotectores , Almacenamiento de Alimentos , Congelación , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Simulación de Dinámica Molecular

RESUMEN

Bacterial endophytes with capacity to promote plant growth and improve plant tolerance against biotic and abiotic stresses have importance in agricultural practice and phytoremediation. A plant growth-promoting endophyte named Klebsiella sp. LTGPAF-6F, which was isolated from the roots of the desert plant Alhagi sparsifolia in north-west China, exhibits the ability to enhance the growth of wheat under drought stress. The complete genome sequence of this strain consists of one circular chromosome and two circular plasmids. From the genome, we identified genes related to the plant growth promotion and stress tolerance, such as nitrogen fixation, production of indole-3-acetic acid, acetoin, 2,3-butanediol, spermidine and trehalose. This genome sequence provides a basis for understanding the beneficial interactions between LTGPAF-6F and host plants, and will facilitate its applications as biotechnological agents in agriculture.

Asunto(s)

Fabaceae/microbiología , Genoma Bacteriano , Klebsiella/aislamiento & purificación , Análisis de Secuencia de ADN/métodos , Composición de Base , Mapeo Cromosómico , Sequías , Klebsiella/genética , Klebsiella/fisiología , Filogenia

RESUMEN

In this study, the natural deep eutectic solvents (NADESs) based on trehalose and choline chloride have been prepared to enhance the protein thermostability. The results of fourier transform infrared spectroscopy and (1)H nuclear magnetic resonance spectroscopy suggested that there were intensive hydrogen-bonding interactions between trehalose and choline chloride in TCCL3-DES and TCCL3-DES75. The physicochemical properties of TCCL3-DES and TCCL3-DES75 were investigated in the temperature range of 293.15-363.15K. Our results revealed that the thermostability of lysozyme, a model protein used in this study was dramatically increased in TCCL3-DES75, as evidenced by the disappearance of the denaturing peak from their Differential Scanning Calorimetry (DSC) traces. The results of circular dichroism (CD) experiments further demonstrated that the lysozyme in TCCL3-DES75 unfolded partially at 90°C and recovered to the initial structure at 20°C. The study suggests that TCCL3-DES75 might be a potential solvent for stabilizing proteins.

Asunto(s)

Fenómenos Químicos , Solventes/química , Trehalosa/química , Rastreo Diferencial de Calorimetría , Colina/química , Dicroismo Circular , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Estructura Molecular , Muramidasa , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

For direct intramacrophagic antitubercular therapy, pulmonary administration through Dry Powder Inhaler (DPI) devices is a reasonable option. For the achievement of efficacious aerosolisation, rifampicin-loaded Solid Lipid Nanoparticle assemblies (SLNas) were developed using the melt emulsifying technique followed by freeze-drying. Indeed, this drying method can cause freezing or drying stresses compromising powder respirability. It is the aim of this research to offer novel information regarding pre-freezing variables. These included type and concentration of cryoprotectants, pre-freezing temperature, and nanoparticle concentration in the suspension. In particular, the effects of such variables were observed at two main levels. First of all, on SLNas characteristics - i.e., size, polydispersity index, zeta-potential, circularity, density, and drug loading. Secondly, on powder respirability, taking into account aerodynamic diameter, emitted dose, and respirable fraction. Considering the complexity of the factors involved in a successful respirable powder, a Design of Experiments (DoE) approach was adopted as a statistical tool for evaluating the effect of pre-freezing conditions. Interestingly, the most favourable impact on powder respirability was exerted by quick-freezing combined with a certain grade of sample dilution before the pre-freezing step without the use of cryoprotectants. In such conditions, a very high SLNas respirable fraction (>50%) was achieved, along with acceptable yields in the final dry powder as well as a reduction of powder mass to be introduced into DPI capsules with benefits in terms of administered drug dose feasibility.

Asunto(s)

Antituberculosos/química , Inhaladores de Polvo Seco/métodos , Lípidos/química , Nanopartículas/química , Administración por Inhalación , Antituberculosos/administración & dosificación , Liberación de Fármacos , Liofilización/métodos , Lípidos/administración & dosificación , Nanopartículas/administración & dosificación , Polvos

RESUMEN

The aim of this work was to use annealing as a tool to optimize the lyophilization cycle by decreasing its duration time, and simultaneously preserve the stability of poly(lactic-co-glycolic acid) nanoparticles and with upmost importance, maintain the structural stability of loaded insulin, used as model therapeutic protein. The contribution of a cryoprotectant in this preservation process was also evaluated. Insulin-loaded nanoparticles co-encapsulated with and without trehalose as cryoprotectant were produced, resulting in a particle size of about 250-300 nm, a PdI around 0.25 and a zeta potential in the range of -20 to -24 mV. The insulin association efficiency was higher than 90%, and the loading capacity was of 11-12%. The use of annealing allowed the decrease of duration time of primary drying in about 38%, representing a global decrease of lyophilization time of around 26%. The residual moisture content of all lyophilizates was around 1%, and the reconstitution of lyophilizates obtained using annealing was even faster than those without annealing. The co-encapsulated trehalose better preserved the nanoparticle size throughout the lyophilization process using annealing, compared to formulation containing no cryoprotectant. Fourier-transform infrared spectroscopy showed that the trehalose-containing nanoparticles presented higher insulin structural maintenance, compared to nanoparticles without cryoprotectant, presenting an insulin structural maintenance of 85.3 ± 0.7% and 86.0 ± 1.0% for annealing and no annealing, respectively. This formulation also presented the closest structural similarity with native insulin. Interestingly, the structural features of insulin loaded into nanoparticles upon lyophilization with and without annealing were practically identical, showing that annealing had no detrimental effect in insulin structure. Circular dichroism and fluorescence spectroscopy confirmed these results. Overall, this work gave rise to the importance of annealing in decreasing the duration time of lyophilization of protein-loaded poly(lactic-co-glycolic acid) nanoparticles, and simultaneously ensuring the stability of the carrier and loaded protein.

Asunto(s)

Portadores de Fármacos/química , Insulina/química , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Química Farmacéutica , Dicroismo Circular , Crioprotectores/química , Liofilización , Microscopía Electrónica de Rastreo , Nanopartículas/ultraestructura , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Estabilidad Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Trehalosa/química

RESUMEN

The hydration parameter h was obtained from the viscosity B-coefficients and the partial molar volume of solute, V2, for various sugars and urea in aqueous solutions. The parameter h showed a good correlation with the parameter α, determined from the activity coefficient of water, representing the solute-solvent interaction. The parameter h also showed a good correlation with the number of equatorial-OH groups (e-OH) for sugars, suggesting that the sugar molecules with the higher e-OH fit more to the water-structure. From the temperature dependence of the parameter h (dh/dT), the negative dh/dT for sugars suggested their water-structure making activity while the positive dh/dT for urea corresponded to its structure breaking effect. From the Arrhenius plot, the activation energy for h, Ea, was determined to be as low as 10 kJ/mol for disaccharides suggesting the stable hydration structure. The Ea increased with a decrease in molecular weight for sugars.

Asunto(s)

Carbohidratos/química , Solventes/química , Soluciones , Viscosidad

RESUMEN

The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants with insulin into PLGA nanoparticles, on the stability of the protein and nanoparticles upon lyophilization. Different lyoprotectants were used, namely trehalose, glucose, sucrose, fructose and sorbitol at 10% (w/v). Insulin-loaded PLGA nanoparticles with co-encapsulated lyoprotectants achieved a mean particle size of 386-466nm, and a zeta potential ranging between -34 and -38mV, dependent on the lyoprotectant used. Formulations had association efficiencies and loading capacities of 85-91% and 10-12%, respectively. The lyophilization process increased the colloidal stability of nanoparticles, and maintained their spherical shape and smooth surface, particularly in presence of lyoprotectants. XRPD revealed that the lyophilizates of nanoparticles with co-encapsulated lyoprotectants were amorphous, whereas formulations with externally added lyoprotectants, except trehalose, showed crystallinity. FTIR assessment showed that co-encapsulating lyoprotectants better preserved insulin structure upon lyophilization with a spectral area overlap of 82-87%, compared to only 72% in lyoprotectant absence. These results were confirmed by circular dichroism spectroscopy. Surprisingly, the simultaneous co-encapsulation and addition of lyoprotectants was detrimental to protein stabilization. The insulin in vitro release studies demonstrated that formulations with co-encapsulated trehalose, glucose, sucrose, fructose and sorbitol achieved 83%, 69%, 70%, 77% and 74%, respectively after 48h. In contrast, formulations added with those lyoprotectants prior lyophilization showed a lower release rate not higher than 60% after 48h. This work gives rise to a different promising strategy of co-encapsulating lyoprotectants and therapeutic proteins, to better stabilize protein structure upon lyophilization.

Asunto(s)

Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Dicroismo Circular , Estabilidad de Medicamentos , Liofilización , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Estructura Secundaria de Proteína , Solubilidad , Difracción de Rayos X

RESUMEN

Pulmonary drug delivery system facilitates local instillation of anticancer drugs to lungs which has proven to be pioneering approach for treatment of lung cancer. This approach led the groundwork for delivering liposomal formulation directly to lungs. Gemcitabine-HCl is currently considered as most effective drug for management of lung cancer. However, its application is limited owing to its metabolism by enzymes present in plasma resulting in reduced efficacy and higher toxicity. In present study, lyophilisation technique was used to convert liposomes into dry powder inhaler, which was formulated using emulsification solvent evaporation technique. The physicochemical properties including size, morphology, entrapment efficiency, loading efficiency etc. of formulated liposomes were evaluated. The prepared liposomal DPI (LDPI) formulations were then examined for solid state characteristics and aerosol performance using cascade impactor. From all the formulations prepared, the LDPI formulated using trehalose as cryoprotectant presented required properties along with desirable deposition pattern. Finally, the optimized formulation was selected for in vitro cell line studies; in vivo studies and stability study. This formulated inhalable particles offers a promising approach for the management of lung cancer through regional chemotherapy.

Asunto(s)

Desoxicitidina/análogos & derivados , Aerosoles , Línea Celular Tumoral , Química Farmacéutica , Desoxicitidina/administración & dosificación , Desoxicitidina/química , Desoxicitidina/farmacocinética , Desoxicitidina/toxicidad , Estabilidad de Medicamentos , Inhaladores de Polvo Seco , Humanos , Liposomas , Solubilidad , Gemcitabina

RESUMEN

Oral drug delivery is the most preferred route for patients; however, the low solubility of drugs and the resultant poor absorption compromise the benefits of oral administration. On the other hand, for years, the overwhelmingly accepted mechanism for enhanced oral absorption using lipid nanocarriers was based on the process of lipid digestion and drug solubilization in the small intestine. Few reports indicated that other bypass pathways are involved in drug absorption in the gastrointestinal tract (GIT) for oral delivery of nanocarriers. Herein, we report a new nanoemulsion system with a denatured globular protein with a diameter of 30 nm, soybean protein isolates (SPI), and bile salt as emulsifiers, aiming to enhance the absorption of insoluble drugs and explore other pathways for absorption. A BCS class II drug, fenofibrate (FB), was used as the model drug. The SPI and bile salt-coated Ns with a diameter of approximately 150 nm were prepared via a high-pressure homogenizing procedure. Interestingly, the present Ns could be converted to solid dosage form using fluid-bed coating technology, maintaining a nanoscale size. Most importantly, in a model of in situ rat intestinal perfusion, Ns could penetrate across the intestinal epithelial barrier into the systemic circulation and then obtain biodistribution into other tissues. In addition, Ns significantly improved FB oral absorption, exhibited as a greater than 2- and 2.5-fold increase in Cmax and AUC0-t, respectively, compared to the suspension formulation. Overall, the present Ns are promising nanocarriers for the oral delivery of insoluble drugs, and the penetration of intact Ns across the GIT barrier into systemic circulation may be a new strategy for improved drug absorption with the use of nanocarriers.

Asunto(s)

Ácidos y Sales Biliares/administración & dosificación , Ácidos y Sales Biliares/química , Portadores de Fármacos/química , Fenofibrato/sangre , Fenofibrato/farmacocinética , Absorción Intestinal , Nanopartículas/administración & dosificación , Proteínas de Soja/química , Administración Oral , Animales , Ácidos y Sales Biliares/farmacocinética , Disponibilidad Biológica , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Química Farmacéutica , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/farmacocinética , Emulsiones/administración & dosificación , Emulsiones/efectos adversos , Emulsiones/química , Emulsiones/farmacocinética , Fenofibrato/administración & dosificación , Fenofibrato/química , Humanos , Nanopartículas/química , Nanopartículas/ultraestructura , Tamaño de la Partícula , Desnaturalización Proteica , Ratas , Solubilidad , Proteínas de Soja/administración & dosificación , Proteínas de Soja/farmacocinética , Suspensiones/farmacocinética , Distribución Tisular , Agua/química