Empowering Naringin's Anti-Inflammatory Effects through Nanoencapsulation.

Abundant in citrus fruits, naringin (NAR) is a flavonoid that has a wide spectrum of beneficial health effects, including its anti-inflammatory activity. However, its use in the clinic is limited due to extensive phase I and II first-pass metabolism, which limits its bioavailability. Thus, lipid nanoparticles (LNPs) were used to protect and concentrate NAR in inflamed issues, to enhance its anti-inflammatory effects. To target LNPs to the CD44 receptor, overexpressed in activated macrophages, functionalization with hyaluronic acid (HA) was performed. The formulation with NAR and HA on the surface (NAR@NPsHA) has a size below 200 nm, a polydispersity around 0.245, a loading capacity of nearly 10%, and a zeta potential of about 10 mV. In vitro studies show the controlled release of NAR along the gastrointestinal tract, high cytocompatibility (L929 and THP-1 cell lines), and low hemolytic activity. It was also shown that the developed LNPs can regulate inflammatory mediators. In fact, NAR@NPsHA were able to decrease TNF-α and CCL-3 markers expression by 80 and 90% and manage to inhibit the effects of LPS by around 66% for IL-1ß and around 45% for IL-6. Overall, the developed LNPs may represent an efficient drug delivery system with an enhanced anti-inflammatory effect.

Squid meal and shrimp hydrolysate as novel protein sources for dog food.

The world's growing pet population is raising sustainability and environmental concerns for the petfood industry. Protein-rich marine by-products might contribute to mitigating negative environmental effects, decreasing waste, and improving economic efficiency. The present study evaluated two marine by-products, squid meal and shrimp hydrolysate, as novel protein sources for dog feeding. Along with the analysis of chemical composition and antioxidant activity, palatability was evaluated by comparing a commercial diet (basal diet) and diets with the inclusion of 150 g kg-1 of squid meal or shrimp hydrolysate using 12 Beagle dogs (2.2 ± 0.03 years). Two in vivo digestibility trials were conducted with six dogs, three experimental periods (10 days each) and three dietary inclusion levels (50, 100 and 150 g kg-1) of squid meal or shrimp hydrolysate in place of the basal diet to evaluate effects of inclusion level on apparent total tract digestibility (ATTD), metabolizable energy content, fecal characteristics, metabolites, and microbiota. Both protein sources presented higher protein and methionine contents than ingredients traditionally used in dog food formulation. Shrimp hydrolysate showed higher antioxidant activity than squid meal. First approach and taste were not affected by the inclusion of protein sources, but animals showed a preference for the basal diet. Effects on nutrient intake reflected the chemical composition of diets, and fecal output and characteristics were not affected by the increasing inclusion levels of both protein sources. The higher ATTD of dry matter, most nutrients and energy of diets with the inclusion of both by-products when compared to the basal diet, suggests their potential to be included in highly digestible diets for dogs. Although not affected by the inclusion level of protein sources, when compared to the basal diet, the inclusion of squid meal decreased butyrate concentration and shrimp hydrolysate increased all volatile fatty acids, except butyrate. Fecal microbiota was not affected by squid meal inclusion, whereas inclusion levels of shrimp hydrolysate significantly affected abundances of Oscillosperaceae (UCG-005), Firmicutes and Lactobacillus. Overall, results suggest that squid meal and shrimp hydrolysate constitute novel and promising protein sources for dog food, but further research is needed to fully evaluate their functional value.

Nutritional Composition and Untargeted Metabolomics Reveal the Potential of Tetradesmus obliquus, Chlorella vulgaris and Nannochloropsis oceanica as Valuable Nutrient Sources for Dogs.

The growing pet population is questioning the sustainability of the pet food system. Although microalgae may constitute a more sustainable food resource, the assessment of their potential for canine diets is almost non-existent. The present study aimed to evaluate the potential of three microalgae species (Tetradesmus obliquus, Chlorella vulgaris and Nannochloropsis oceanica) grown locally in industrial photobioreactors as alternative food resources for dogs. A detailed characterization of their nutritional composition and metabolomic profile was carried out and related to the nutritional requirements of dogs. Overall, the essential amino acid content exceeded the amounts required for dogs at all life stages, except methionine and cysteine. The three microalgae were deficient in linoleic acid, N. oceanica presented a linolenic acid content below requirements and T. obliquus and C. vulgaris were deficient in arachidonic and eicosapentaenoic acids. The fiber was mainly composed of insoluble dietary fiber. The mineral profile varied greatly with the microalgae species, demonstrating their different potential for dog feeding. Untargeted metabolomics highlighted glycolipids, glycerolipids and phospholipids as the most discriminating compounds between microalgae species. Overall, the results support the potential of T. obliquus, C. vulgaris and N. oceanica as valuable macro- and micro-nutrients sources for dog feeding.

Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers.

The evaluation of encapsulation efficiency is a regulatory requirement for the characterization of drug delivery systems. However, the difficulties in efficiently separating nanomedicines from the free drug may compromise the achievement of accurate determinations. Herein, ultrafiltration was exploited as a separative strategy towards the evaluation of methotrexate (MTX) encapsulation efficiency in nanostructured lipid carriers and polymeric nanoparticles. The effect of experimental conditions such as pH and the amount of surfactant present in the ultrafiltration media was addressed aiming at the selection of suitable conditions for the effective purification of nanocarriers. MTX-loaded nanoparticles were then submitted to ultrafiltration and the portions remaining in the upper compartment of the filtering device and in the ultrafiltrate were collected and analyzed by HPLC-UV using a reversed-phase (C18) monolithic column. A short centrifugation time (5 min) was suitable for establishing the amount of encapsulated MTX in nanostructured lipid carriers, based on the assumption that the free MTX concentration was the same in the upper compartment and in the ultrafiltrate. The defined conditions allowed the efficient separation of nanocarriers from the free drug, with recoveries of >85% even when nanoparticles were present in cell culture media and in pig skin surrogate from permeation assays.

Ecotoxicological equilibria of triclosan in Microtox, XenoScreen YES/YAS, Caco2, HEPG2 and liposomal systems are affected by the occurrence of other pharmaceutical and personal care emerging contaminants.

Contaminants of emerging concern may be considered as any chemicals or factors whose unintended continuous release and persistence in the environment may lead to any observable undesirable response of living beings. Still not much is known on reciprocal toxicological impact of given chemicals when present in binary or more complex mixtures. In this work, an attempt was thus undertaken to study the impact of butylparaben, methylparaben and diclofenac on toxicological behavior and properties of triclosan (at varying concentration levels) with respect to Microtox, XenoScreen YES/YAS, Caco-2, HEPG2, and liposomal systems. Having performed analytical and biological studies modeling was done using two modeling approaches, viz., concentration addition (CA) and independent action (IA) at three concentration levels of each chemical studied. The effect of the highest concentration of triclosan studied was impacted by even small amounts of methylparaben and butylparaben in Microtox while diclofenac preferably affected triclosan activity at its lowest concentration level (with CA model). Estrogenic agonistic properties of triclosan were severely impacted by both parabens in an antagonistic way; diclofenac showed in all cases underestimation or synergy at the lowest triclosan concentration studied. Estrogenic antagonistic activity of triclosan was also slightly affected by parabens and by diclofenac in binary mixtures, showing overestimation and antagonist effects. HepG2 cells appeared to be the most resistant to the toxic effect of the mixtures at the concentrations tested and no significant proof of synergy or antagonism could be detected with the MTT assay. The liposome assays on the mixtures followed the same trends obtained with the MTT assay with Caco-2 cells, confirming the validity of the in vitro model used in this research. As studies on emerging contaminants mixtures toxicity are still scarce, research presented here constitute an important part in confirming utility and versatility of emerging contaminants modeling in environmental toxicology.

Novel hydrogels based on yeast chitin-glucan complex: Characterization and safety assessment.

Chitin-glucan complex (CGC) was used for the first time for the preparation of hydrogels. Alkali solvent systems, NaOH and KOH solutions, either at 1 or 5 mol/L, were used for CGC dissolution using a freeze-thaw procedure (freezing at -20 °C and thawing at room temperature; four cycles). The CGC solutions thus obtained were subjected to dialysis that induced the spontaneous gelation of the biopolymer, yielding translucid hydrogels with a yellowish coloration. Although all CGC hydrogels exhibited porous microstructures, high water content (above 97%) and good mechanical properties, their morphology, viscoelastic properties and texture were influenced by the type of solvent system used for CGC dissolution, as well as by their ionic strength. The K-based hydrogels presented a less compact network with larger pores and exhibited lower elastic properties. The Na-based hydrogels, on the other hand, exhibited a denser structure with smaller pores and a stiffer gel structure. These results show that it is possible to prepare CGC hydrogels with differing characteristics that can be suitable for different applications. Furthermore, all hydrogels were non-cytotoxic towards L929 fibroblasts and HaCaT keratinocytes. This study demonstrates CGC can be used to prepare biocompatible hydrogels with properties render them promising biomaterials.

Chromatographic method for the simultaneous quantification of dapsone and clofazimine in nanoformulations.

The low bioavailability and nonspecific distribution of dapsone and clofazimine, commonly applied in combination for the treatment of leprosy, can produce toxic effects. Nanotechnological approaches enhance the delivery of these drugs. Therefore, a high-performance liquid chromatography method was developed for the simultaneous determination of dapsone and clofazimine loaded in nanoformulations for quality control purposes. Chromatographic separation was achieved on a reversed-phase Kinetex core-shell C18 column, followed by spectrophotometric detection at 280 nm. Considering the different physicochemical properties of dapsone and clofazimine, elution was performed in gradient mode using an aqueous acetate buffer (50 mmol/L, pH 4.8) and an increasing acetonitrile content from 27 to 63% v/v at a flow rate of 1.0 mL/min with retention times of 6.2 and 14.0 min, respectively. The method was validated according to the European Medicines Agency guideline and it was found to be specific, accurate (99.6-114.0%), and precise for intra- (RSD ≤ 1.8%) and interday assays (RSD ≤ 12.5%). Both drugs showed stability after 24 h at room temperature and over three freeze-thaw cycles with recoveries ≥86.2%. Low temperature (4°C) in the autosampler caused the precipitation of clofazimine and must be avoided. The validated method was successfully applied in the quantification of both drugs in nanoformulations.

Multiple Lipid Nanoparticles (MLN), a New Generation of Lipid Nanoparticles for Drug Delivery Systems: Lamivudine-MLN Experimental Design.

PURPOSE: An optimized methodology for the development of a new generation of lipid nanoparticles, the multiple lipid nanoparticles (MLN) is described. MLN have characteristics between nanostructured lipid carriers (NLC) and multiple emulsions (W/O/W), but without the outer aqueous phase. METHODS: The production is based on a hot homogenization method combined with high shear and ultrasonication. The antiretroviral agent lamivudine (3TC), was loaded in the MLN. For comparison purposes, NLC-3TC formulation was also developed and physico-chemically characterized by the same parameters as MLN-3TC. The development and optimization of MLN and NLC formulations were supported by a Quality by Design (QbD) approach. RESULTS: The MLN-3TC formulation exhibited a size of about 450 nm, polydispersity <0.3 and negative zeta potential > -20 mV. Furthermore, the morphology assessed by TEM showed a structure with multiples aqueous vacuoles. MLN-3TC was physically stable for at least 45 days, had low cytotoxicity and drug release studies showed a sustained and controlled release of 3TC under gastric and plasma-simulated conditions (at pH 7.4 for about 45 h). CONCLUSIONS: The optimized formulations present suitable profiles for oral administration. Overall, the results reveal that MLN present higher loading capacity and storage stability than NLC.

Development and validation of HPLC method with fluorometric detection for quantification of bisnaphthalimidopropyldiaminooctane in animal tissues following administration in polymeric nanoparticles.

A simple, sensitive and specific high-performance liquid chromatography method for the quantification of bisnaphthalimidopropyldiaminooctane (BNIPDaoct), a potent anti-Leishmania compound, incorporated into poly(d,l-lactide-co-glycolic acid) (PLGA) nanoparticles was developed and validated toward bioanalysis application. Biological tissue extracts were injected into a reversed-phase monolithic column coupled to a fluorimetric detector (λexc=234nm, λem=394nm), using isocratic elution with aqueous buffer (acetic acid/acetate 0.10M, pH 4.5, 0.010M octanesulfonic acid) and acetonitrile, 60:40 (v/v) at a flow rate of 1.5mLmin(-1). The run time was 6min, with a BNIPDaoct retention time of 3.3min. Calibration curves were linear for BNIPDaoct concentrations ranging from 0.002 to 0.100µM. Matrix effects were observed and calibration curves were performed using the different organ (spleen, liver, kidney, heart and lung) extracts. The method was found to be specific, accurate (97.3-106.8% of nominal values) and precise for intra-day (RSD<1.9%) and inter-day assays (RSD<7.2%) in all matrices. Stability studies showed that BNIPDaoct was stable in all matrices after standing for 24h at room temperature (20°C) or in the autosampler, and after three freeze-thaw cycles. Mean recoveries of BNIPDaoct spiked in mice organs were >88.4%. The LOD and LOQ for biological matrices were ≤0.8 and ≤1.8nM, respectively, corresponding to values ≤4 and ≤9nmolg(-1) in mice organs. The method developed was successfully applied to biodistribution assessment following intravenous administration of BNIPDaoct in solution or incorporated in PLGA nanoparticles.

Benzodiazepine-mediated structural changes in the multidrug transporter P-glycoprotein: an intrinsic fluorescence quenching analysis.

P-glycoprotein expressed in Pichia pastoris was used to study the drug binding sites of different benzodiazepines. The effect of bromazepam, chlordiazepoxide, diazepam and flurazepam on P-glycoprotein structure was investigated by measuring the intrinsic fluorescence of the transporter tryptophan residues. Purified mouse mdr1a transporter in mixed micelles of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid and 1,2-dimiristoyl-sn-glycerol-3-phosphocholine emitted fluorescence at 340 nm indicative of the fluorophores in a relatively apolar environment. Acrylamide and iodide ion were used as collisional quenchers toward distinct regions of the transporter, the protein and the interface protein-surface, respectively. Binding of ATP induced conformational changes at the protein surface level in accordance with the location of the nucleotide binding sites. Bromazepam interaction with the transporter was located at the protein-surface interface, diazepam at the membrane region and chlordiazepoxide at the protein surface. Only the flurazepam interaction site was not detected by the quenchers used. All benzodiazepines were able to elicit reorientation of the protein fluorophores on the P-glycoprotein-ATP complex.

Flurazepam inhibits the P-glycoprotein transport function: an insight to revert multidrug-resistance phenotype.

P-glycoprotein mediated drug transport may lead to a multidrug resistance phenotype often associated with a poor response to the successful treatment of a variety of human disorders. Several agents have been found to modulate P-glycoprotein drug resistance, most probably by blocking its transport function. The aim of this study was to examine the effects of some benzodiazepines (bromazepam, chlordiazepoxide, diazepam and flurazepam) able to bind to P-glycoprotein in proteoliposomes on its transport function and ATPase activity in the human cancer cell line, KB-V1. The toxicity of the benzodiazepines drugs towards KB-V1 cells was first evaluated and the non toxic drugs concentrations were used to assess the drug efflux and the ATPase activity. Using the flow cytometry approach, the accumulation and efflux of daunorubicin were followed by measuring the daunorubicin associated geometric mean fluorescence intensity. Vanadate was employed as a comparative inhibitory compound. Flurazepam was able to inhibit the daunorubicin efflux in 80%. ATPase activity determined by a colorimetric assay revealed that flurazepam inhibits the P-glycoprotein enzymatic activity, indicating coupling between drug transport and ATP hydrolysis. Bromazepam, chlordiazepoxide and diazepam behaved as activators of the P-glycoprotein ATPase activity, suggesting a role as transported substrates and did not interfere in the daunorubicin transport.

Sensitivity of P-glycoprotein tryptophan residues to benzodiazepines and ATP interaction.

Plasma membrane P-glycoprotein is a member of the ATP-binding cassette family of membrane transporters. In the present study tryptophan intrinsic fluorescence was used to understand the P-glycoprotein response to three benzodiazepines (bromazepam, chlordiazepoxide and flurazepam) in the presence and absence of ATP. Fluorescence emission spectra showed a red shift on the maximal emission wavelength upon interaction of P-glycoprotein with all benzodiazepines. Benzodiazepine association with nucleotide-bound P-glycoprotein also showed this trend and the quenching profile was attributed to a sphere-of-action model, for static fluorescence. Furthermore, quenching data of benzodiazepine-bound P-glycoprotein with ATP were concentration dependent and saturable, indicating that nucleotide binds to P-glycoprotein whether drug is present or not. These results seems in agreement with the proposal of the ATP-switch model by Higgins and Linton, where substrate binding to the transporters initiates the transport cycle by increasing the ATP binding affinity.

Biodegradation of p-chlorophenol by a microalgae consortium.

An aquatic community was recovered from a waste discharge container fed with several aromatic pollutants. After 3 months of selective enrichment with p-chlorophenol and p-nitrophenol, two microalgae species, Chlorella vulgaris and Coenochloris pyrenoidosa, were recovered from the microbial consortium. As an axenic culture, this microalgae consortium was able to remove p-chlorophenol under different photo-regimes. Cultures grown under a 24h light regime were capable of biodegrading 50mg l(-1) of p-chlorophenol within 5 days. Addition of zeolite, an adsorbing material, did not improve the p-chlorophenol removal. However, when p-chlorophenol at 150mgl(-1) was fed to the culture supplemented with zeolite, the growth rate of the consortium improved, but the lag phase was longer (16 against 14 days in the absence of zeolite).