Ethyl Cellulose-Core, OSA Starch-Shell Electrosprayed Microcapsules Enhance the Oxidative Stability of Loaded Fish Oil.

The encapsulation and the oxidative stability of cod liver fish oil (CLO) within coaxial electrosprayed (ethyl cellulose/CLO) core-(octenyl succinic anhydride, OSA-modified starch) shell, and monoaxial electrosprayed ethyl cellulose/CLO microcapsules were investigated. Core-shell (H-ECLO) and monoaxial (ECLO) electrosprayed microcapsules with an average diameter of 2.8 ± 1.8 µm, and 2.2 ± 1.4 µm, respectively, were produced. Confocal microscopy confirmed not only the core-shell structure of the H-ECLO microcapsules, but also the location of the CLO in the core. However, for the ECLO microcapsules, the CLO was distributed on the microcapsules' surface, as also confirmed by Raman spectroscopy. Atomic force microscopy showed that the average surface adhesion of the H-ECLO microcapsules was significantly lower (5.41 ± 0.31 nN) than ECLO microcapsules (18.18 ± 1.07 nN), while the H-ECLO microcapsules showed a remarkably higher Young's modulus (33.84 ± 4.36 MPa) than the ECLO microcapsules (6.64 ± 0.84 MPa). Differential scanning calorimetry results confirmed that the H-ECLO microcapsules enhanced the oxidative stability of encapsulated CLO by about 15 times, in comparison to non-encapsulated oil, mainly by preventing the presence of the fish oil at the surface of the microcapsules, while ECLO microcapsules enhanced the oxidative stability of CLO about 2.9 times due to the hydrophobic interactions of the oil and ethyl cellulose. Furthermore, the finite element method was also used to evaluate the electric field strength distribution, which was substantially higher in the vicinity of the collector and lower in the proximity of the nozzle when the coaxial electrospray process was employed in comparison to the monoaxial process.

Carbohydrate Core-Shell Electrosprayed Microcapsules for Enhanced Oxidative Stability of Vitamin A Palmitate.

Vitamin A is an essential micronutrient that is readily oxidized. In this study, the encapsulation of vitamin A palmitate (AP) within a core-shell carbohydrate matrix by co-axial electrospray and its oxidative stability was evaluated. The electrosprayed core-shell microcapsules consisted of a shell of octenyl succinic anhydride (OSA) modified corn starch, maltose (Hi-Cap), and a core of ethyl cellulose-AP (average diameter of about 3.7 µm). The effect of different compounds (digestion-resistant maltodextrin, soy protein hydrolysate, casein protein hydrolysate, and lecithin) added to the base core-shell matrix formulation on the oxidative stability of AP was investigated. The oxidative stability of AP was evaluated using isothermal and non-isothermal differential scanning calorimetry (DSC), and Raman and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy methods. The core-shell carbohydrate matrix minimizes the amount of AP present at the microparticle surface, thus protecting AP from oxidation. Furthermore, the most effective oxidation protection was achieved when casein protein hydrolysate was added to the core of the microcapsule due to hydrophobic and hydrogen bond interactions with AP and by the resistant maltodextrin in the shell, which acted as a filler. The utilization of ethanol as a solvent for the dispersion of the core compounds increased the hydrophobicity of the hydrolyzed proteins and contributed to the enhancement of their antioxidant ability. Both the carbohydrate core-shell microcapsule prepared by co-axial electrospray and the addition of oxidation protection compounds enhance the oxidative stability of the encapsulated AP.

Enhanced electric field and charge polarity modulate the microencapsulation and stability of electrosprayed probiotic cells (Streptococcus thermophilus, ST44).

The effect of the polarity of the direct current electric field on the "organization" of Streptococcus thermophilus (ST44) probiotic cells within electrosprayed maltodextrin microcapsules was investigated. The generated electrostatic forces between the negatively surface-charged probiotic cells and the applied negative polarity on the electrospray nozzle, allowed to control the location of the cells towards the core of the electrosprayed microcapsules. This "organization" of the cells increased the evaporation of the solvent (water) and successively the glass transition temperature (Tg) of the electrosprayed microcapsules. Moreover, the utilization of auxiliary ring-shaped electrodes between the nozzle and the collector, enhanced the electric field strength and contributed further to the increase of the Tg. Numerical simulation, through Finite Element Method (FEM), shed light to the effects of the additional ring-electrode on the electric field strength, potential distribution, and controlled deposition of the capsules on the collector. Furthermore, when the cells were located at the core of the microcapsules their viability was significantly improved for up to 2 weeks of storage at 25 °C and 35% RH, compared to the case where the probiotics were distributed towards the surface. Overall, this study reports a method to manipulate the encapsulation of the surface charged probiotic cells within electrosprayed microcapsules, utilizing the polarity of the electric field and additional ring-electrodes.

Biotechnologically produced chitosans with nonrandom acetylation patterns differ from conventional chitosans in properties and activities.

Chitosans are versatile biopolymers with multiple biological activities and potential applications. They are linear copolymers of glucosamine and N-acetylglucosamine defined by their degree of polymerisation (DP), fraction of acetylation (FA), and pattern of acetylation (PA). Technical chitosans produced chemically from chitin possess defined DP and FA but random PA, while enzymatically produced natural chitosans probably have non-random PA. This natural process has not been replicated using biotechnology because chitin de-N-acetylases do not efficiently deacetylate crystalline chitin. Here, we show that such enzymes can partially N-acetylate fully deacetylated chitosan in the presence of excess acetate, yielding chitosans with FA up to 0.7 and an enzyme-dependent non-random PA. The biotech chitosans differ from technical chitosans both in terms of physicochemical and nanoscale solution properties and biological activities. As with synthetic block co-polymers, controlling the distribution of building blocks within the biopolymer chain will open a new dimension of chitosan research and exploitation.

Adjustable polysaccharides-proteins films made of aqueous wheat proteins and alginate solutions.

Large amount of wheat proteins by-products are produced during wheat starch manufacture. This work aimed to develop edible films of cast aqueous wheat proteins (WP) and alginate (Al) solutions. The investigation of the microstructure of Al/WP films revealed a more compacted cross-section and homogeneous surface, comparatively to Al films. Those properties could be modified with the increase of WP concentration from 4 to 8 % w/v, as result of electrostatic interactions between WP and Al. Furthermore, the incorporation of WP provided UltraViolet-blocking behaviour (4-fold decrease in the Ultra-Violet-B region). Additionally, the incorporation of WP in the films reduced the water solubility of the Al films. It was also found that by incorporating different amounts of WP the mechanical and Water Vapor Transmission rate (WVTR) properties could also be modified, so the film composition could be adjusted to suit different types of foods and applications (e.g. coatings and packaging).

Cyclic Citrullinated Peptide Aptamer Treatment Attenuates Collagen-Induced Arthritis.

We describe the study of a novel aptamer-based candidate for treatment of seropositive rheumatoid arthritis. The candidate is a nanoparticle-formulated cyclic citrullinated peptide aptamer, which targets autoantibodies and/or the immune reactions leading to antibody production. Due to its specificity, the peptide aptamer nanoparticles might not interfere with normal immune functions as seen with other disease-modifying antirheumatic drugs. Over a 3-week course of treatment, joint swelling and arthritis score in collagen-induced rats were significantly decreased compared with animals treated with phosphate-buffered saline, unloaded nanoparticles, or nanoparticles with a noncitrullinated control peptide. The reduction in joint swelling was associated with decreased anticitrullinated peptide autoantibody levels in the blood. Treatment with aptamer nanoparticles also increased interleukin-10 levels. The effect seen with the proposed treatment candidate could be mediated by upregulation of anti-inflammatory mediators and decreased levels of anticitrullinated peptide antibodies.

Pediatric adrenal insufficiency: thirty years experience at a Portuguese hospital.

BACKGROUND: Adrenal insufficiency (AI) is a life-threatening condition caused by an impaired secretion of the adrenal glucocorticoid and mineralocorticoid hormones. It comprises a heterogeneous group of primary, secondary and acquired disorders. Presentation differs according to the child's age, but it usually presents with nonspecific and insidious symptoms and signs. The main purpose of this study was to describe and compare patients with primary or secondary AI. METHODS: Retrospective analysis of all patients with adrenal insufficiency followed at the Pediatric Endocrinology Unit in a tertiary care Portuguese hospital over the last 30 years. Data on family history, age at the first manifestation and at etiological diagnosis, and clinical presentation (symptoms, signs and laboratory evaluation) was gathered for all patients. RESULTS: Twenty-eight patients with AI were included; 67.9% were male, with a median (25th-75th percentile, P25-P75) age of 1 (0.5-36) month at the first presentation. The principal diagnostic categories were panhypopituitarism (42.9%) and congenital adrenal hyperplasia (25%). The most frequent manifestations (75%) were vomiting and weight loss. They were followed for a median (P25-P75) period of 3.5 (0.6-15.5) years. In respect to neurodevelopmental delay and learning difficulties, they were more common in the secondary AI group. CONCLUSIONS: Despite medical advances, the diagnosis and management of AI remains a challenge, particularly in the pediatric population, and clinicians must have a high index of suspicion. An early identification of AI can prevent a potential lethal outcome, which may result from severe cardiovascular and hemodynamic instability.

Brushing Effect on the Properties of Glass Ionomer Cement Modified by Hydroxyapatite Nanoparticles or by Bioactive Glasses.

This study evaluated the physical and mechanical properties of glass ionomer cement (GIC) associated with 5% hydroxyapatite nanoparticles (NPHAps) and 10% bioactive glass (BAG) 45S5 before and after brushing at different storage times. Surface roughness was evaluated using a rugosimeter, Vickers hardness using a microdurometer, and mass variation measured in an analytical balance at 1, 7, 15, 30, and 60 days before and after the brushing test, with the aid of toothbrushing simulator and soft bristle toothbrushes. Nonnormal distribution was observed, and the nonparametric Wilcoxon and Kruskal-Wallis tests followed by Dunn's were performed, with a significance level of 5%. We observed higher values for mass loss on the first day for all groups. The surface roughness was lower in the control and NP groups, 30 days after brushing. Higher values for hardness were found in the control group and lower ones for NP, after brushing. The control and BAG groups presented a decrease in hardness over time. The NP group presented the highest values before brushing, while the control group had the highest values after brushing. The association of NPHPa with the GIC is the most promising combination, since it presented satisfactory values for surface hardness. However, conventional GIC not associated with NPHPa or BAG is still an option, since it is available in the market and the most economically viable option.

Carbon Nanotubes-Potent Carriers for Targeted Drug Delivery in Rheumatoid Arthritis.

Two types of single-walled carbon nanotubes (SWCNTs), HiPco- and carboxyl-SWCNT, are evaluated as drug carriers for the traditional anti-inflammatory drug methotrexate (MTX) and a small interfering RNA (siRNA) targeting NOTCH1 gene. The nanotubes are solubilized by PEGylation and covalently loaded with MTX. The coupling efficiency (CE%) of MTX is 77-79% for HiPco-SWCNT and 71-83% for carboxyl-SWCNT. siRNA is noncovalently attached to the nanotubes with efficiency of 90-97% for HiPco-SWCNT and 87-98% for carboxyl-SWCNT. Through whole body imaging in the second near-infrared window (NIR-II window, 1000-1700 nm), SWCNTs were found to be selectively accumulated in inflamed joints in a serum transfer mouse model. We further investigated the interactions of the siRNA/MTX loaded nanotubes with human blood and mice bone marrow cells. In human blood, both types of unloaded SWCNTs were associated with B cells, monocytes and neutrophils. Interestingly, loading with MTX suppressed SWCNTs targeting specificity to immune cells, especially B cells; in contrast, loading siRNA alone enhanced the targeting specificity. Loading both MTX and siRNA to carboxyl-SWCNT enhanced targeting specificity to neutrophils and monocytes but not B cells. The targeting specificity of SWCNTs can potentially be adjusted by altering the ratio of MTX and siRNA loaded. The combined results show that carbon nanotubes have the potential for delivery of cargo drugs specifically to immune cells involved in rheumatoid arthritis.

Electrosprayed Ethyl Cellulose Core-Shell Microcapsules for the Encapsulation of Probiotics.

Electrosprayed ethyl cellulose core-shell microcapsules were produced for the encapsulation of probiotic Bifidobacterium animalis subsp. lactis (Bifido). Ethyl cellulose (ETC) was used as a shell material with different core compounds (concentrated Bifido, Bifido-maltodextrin and Bifido-glycerol). The core-shell microcapsules have an average diameter between 3 µm and 15 µm depending on the core compounds, with a distinct interface that separates the core and the shell structure. The ETC microcapsules displayed relatively low water activity (aw below 0.20) and relatively high values of viable cells (109-1011 CFU/g), as counted post-encapsulation. The effect of different core compounds on the stability of probiotics cells over time was also investigated. After four weeks at 30 °C and 40% RH the electrospray encapsulated samples containing Bifido-glycerol in the core showed a loss in viable cells of no more than 3 log loss CFU/g, while the non-encapsulated Bifido lost about 7.57 log CFU/g. Overall, these results suggest that the viability of the Bifido probiotics encapsulated within the core-shell ETC electrosprayed capsules can be extended, despite the fact that the shell matrix was prepared using solvents that typically substantially reduce their viability.

Electrohydrodynamic Processing of Potato Protein into Particles and Fibers.

Potato protein particles and fibers were produced using electrohydrodynamic processing (electrospray and electrospinning). The effect of different solvents and protein concentration on the morphology of the potato protein particles and fibers was investigated. Electrosprayed particles with average diameters ranging from 0.3 to 1.4 µm could be obtained using water and mixtures of water: ethanol (9:1) and water:glycerol (9:1). Electrosprayed particles were also obtained using the solvent hexafluoro-2-propanol (HFIP) at a protein concentration of 5% wt/v. For protein concentrations above 10% wt/v, using HFIP, electrospun fibers were produced. The release of vitamin B12, as a model bioactive compound, from potato protein electrospun fibers, was also investigated, demonstrating their potential to be utilized as encapsulation and delivery systems.

Evaluation of thrombocytopenia in patients with non-alcoholic fatty liver disease without cirrhosis.

INTRODUCTION AND OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disorder in western countries. It is often related to metabolic syndrome, presenting an increased risk of advanced liver disease and cardiovascular-related death. In some etiologies of chronic liver disease, thrombocytopenia has been associated not only with advanced stages of fibrosis but also with autoimmune disease. In NAFLD, however, its prevalence and related factors are still unknown. The aim of this study is to evaluate the prevalence of thrombocytopenia in NAFLD patients without cirrhosis and to investigate its related risk factors. PATIENTS AND METHODS: This was a retrospective study carried out in two tertiary hospitals in the South and Southeast regions of Brazil. Patients diagnosed with NAFLD by liver biopsy were included. Those with other causes of liver disease and/or cirrhosis were excluded. For analysis, patients were divided into two groups, with and without thrombocytopenia. Data was analyzed using a significance level of 5%. RESULTS: 441 non-cirrhotic patients with NAFLD (evaluated by liver biopsy) were included in the study. The prevalence of thrombocytopenia was 3.2% (14/441 patients). In the comparative analysis between groups, thrombocytopenia was associated with male sex (p=0.007) and level of hemoglobin (p=0.023). CONCLUSION: Thrombocytopenia is an infrequent event in NAFLD patients without cirrhosis and is related with male sex and higher hemoglobin levels.

Low-Fouling Electrosprayed Hemoglobin Nanoparticles with Antioxidant Protection as Promising Oxygen Carriers.

Despite all the attempts to create advanced hemoglobin (Hb)-based oxygen carriers (HBOCs) employing an encapsulation platform, major challenges including attaining a high Hb loading and long circulation times still need to be overcome. Herein, the fabrication, for the first time, of nanoparticles fully made of Hb (Hb-NPs) employing the electrospray technique is reported. The Hb-NPs are then coated by antioxidant and self-polymerized poly(dopamine) (PDA) to minimize the conversion of Hb into nonfunctional methemoglobin (metHb). The PDA shell is further functionalized with poly(ethylene glycol) (PEG) to achieve stealth properties. The results demonstrate that the as-prepared Hb-NPs are hemo- and biocompatible while offering antioxidant protection and decreasing the formation of metHb. Additionally, decoration with PEG results in decreased protein adsorption onto the Hb-NPs surface, suggesting a prolonged retention time within the body. Finally, the Hb-NPs also preserve the reversible oxygen-binding and releasing properties of Hb. All in all, within this study, a novel HBOCs with high Hb content is fabricated and its potential as an artificial blood substitute is evaluated.

Citrullinated Peptide Epitope Targets Therapeutic Nanoparticles to Human Neutrophils.

Multiple drugs have been proposed for reducing harsh symptoms of human rheumatic diseases. However, a targeted therapy with mild to no side effects is still missing. In this study, we have prepared and tested a series of therapeutic nanoparticles for specific targeting of human neutrophils associated with rheumatoid arthritis. In doing this, a series of citrullinated peptide epitopes derived from human proteins, fibrinogen, vimentin, and histone 3, were screened with regard to specific recognition of neutrophils. The most potent epitope proved to be a mutated fragment of an alpha chain in human fibrinogen. Next, a straightforward synthetic strategy was developed for nanoparticles decorated with this citrullinated peptide epitope and an antisense oligonucleotide targeting disease associated microRNA miR-125b-5p. Our study shows that the nanoparticles specifically recognize neutrophils and knock down miR-125b-5p, with no apparent toxicity to human cells. In contrast to organic dendrimers, chitosan-hyaluronic acid formulations do not activate human innate immune response. Our data proves that the strategy we report herein is effective in developing peptide epitopes for decorating delivery vehicles bearing biological drugs, targeted to a specific cell type.

Electrospinning and electrospraying technologies for food applications.

Electrospinning and electrospraying are versatile techniques for the production of nano- to micro-scale fibers and particles. Over the past 2 decades, significant progresses have been made to advance the fundamental understandings of these electrohydrodynamic processes. Researchers have investigated different polymeric and non-polymeric substrates for producing submicron electrospun/electrosprayed materials of unique morphologies and physicochemical properties. This chapter provides an overview on the basic principles of electrospinning and electrospraying, highlighting the effects of key processing and solution parameters. Electrohydrodynamic phenomena of edible substrates, including polysaccharides (xanthan, alginate, starch, cyclodextrin, pullulan, dextran, modified celluloses, and chitosan), proteins (zein, what gluten, whey protein, soy protein, gelatin, etc.), and phospholipids are reviewed. Selected examples are presented on how ultrafine fibers and particles derived from these substrates are being exploited for food and nutraceutical applications. Finally, the challenges and opportunities of the electrostatic methods are discussed.

Enhanced Transepithelial Permeation of Gallic Acid and (-)-Epigallocatechin Gallate across Human Intestinal Caco-2 Cells Using Electrospun Xanthan Nanofibers.

Electrospun xanthan polysaccharide nanofibers (X) were developed as an encapsulation and delivery system of the poorly absorbed polyphenol compounds, gallic acid (GA) and (-)-epigallocatechin gallate (EGCG). Scanning electron microscopy was used to characterize the electrospun nanofibers, and controlled release studies were performed at pH 6.5 and 7.4 in saline buffer, suggesting that the release of polyphenols from xanthan nanofibers follows a non-Fickian mechanism. Furthermore, the X-GA and X-EGCG nanofibers were incubated with Caco-2 cells, and the cell viability, transepithelial transport, and permeability properties across cell monolayers were investigated. An increase of GA and EGCG permeability was observed when the polyphenols were loaded into xanthan nanofibers, compared to the free compounds. The observed in vitro permeability enhancement of GA and EGCG was induced by the presence of the polysaccharide nanofibers, which successfully inhibited efflux transporters, as well as by tight junctions opening.

In vitro permeability enhancement of curcumin across Caco-2 cells monolayers using electrospun xanthan-chitosan nanofibers.

Xanthan-Chitosan (X-Ch) polysaccharides nanofibers were prepared using electrospinning processing as an encapsulation and delivery system of curcumin (Cu). The X-Ch-Cu nanofibers remained stable in aqueous HBSS medium at pH 6.5 and pH 7.4, mainly due to the ability of oppositely charged xanthan-chitosan polyelectrolytes to form ionically associated electrospun nanofibers. The xanthan-chitosan-curcumin nanofibers were incubated with Caco-2 cells, and the cell viability, transepithelial transport and permeability properties across cell monolayers were investigated. After 24 h of incubation, the exposure of Caco-2 cell monolayers to X-Ch-Cu nanofibers resulted in a cell viability of ∼80%. A 3.4-fold increase of curcumin permeability was observed when the polyphenol was loaded into X-Ch nanofibers, compared to the free curcumin. This increased in vitro transepithelial permeation of curcumin without compromising cellular viability was induced by interactions upon contact between the nanofibers and the Caco-2 cells, leading to the opening of the tight junctions. The results obtained revealed that X-Ch nanofibers can be used for oral delivery applications of poorly water-soluble compounds at the gastrointestinal tract.

Preparation and Characterization of an Oral Vaccine Formulation Using Electrosprayed Chitosan Microparticles.

Chitosan particles loaded with the antigen ovalbumin (OVA) and the adjuvant Quil-A were produced by electrospray, using mixtures of water/ethanol/acetic acid as a solvent. Three different chitosans designed as HMC+70, HMC+85, and HMC+90 (called as 705010, 855010, and 905010) were tested and its efficacy to be used in oral vaccine delivery applications was investigated. The morphology, size, and zeta potential of the produced particles were investigated, together with the encapsulation efficiency and release of OVA from the three chitosan formulations. Moreover, the mucoadhesion and cytotoxicity of the chitosan microparticles was examined. All the three formulations with OVA and Quil-A were in the micrometer size range and had a positive zeta potential between 46 and 75 mV. Furthermore, all the three formulations displayed encapsulation efficiencies above 80% and the release of OVA over a period of 80 h was observed to be between 38 and 47%. None of the developed formulations exhibited high mucoadhesive properties, either cytotoxicity. The formulation prepared with HMC+70, OVA, and Quil-A had the highest stability within 2 h in buffer solution, as measured by dynamic light scattering. The electrosprayed formulation consisting of HMC+70 with OVA and Quil-A showed to be the most promising as an oral vaccine system.

Morphological, Mechanical and Mucoadhesive Properties of Electrospun Chitosan/Phospholipid Hybrid Nanofibers.

This study aimed to develop hybrid electrospun chitosan⁻phospholipid nanofibers and investigate the effect of phospholipid (P) content and chitosans (Ch) molecular weights (Mw) and degree of acetylation (DA), on the morphological, mechanical and mucoadhesive properties of the nanofibers. Electrospun Ch/P nanofibers exhibited a smooth and uniform surface with average diameters ranging from 300 to 1000 nm, as observed by scanning electron microscopy (SEM). The average diameter of the nanofibers was observed to increase with the increase of the Mw and degree of deacetylation of Ch, and phospholipid content. The elastic and adhesive properties of the nanofibers were determined by atomic force microscopy, and displayed higher values for higher Mw and lower DA Ch used. The elastic modulus of electrospun Ch/P hybrid fibers determined for the different conditions tested was found to be in the range of 500 and 1400 MPa. Furthermore, electrospun Ch/P nanofibers displayed mucoadhesive properties expressed by the work of adhesion calculated after the compression of the nanofibers against a section of pig small intestine. Our results showed that the increase in phospholipid content and DA of Ch decrease the work of adhesion, while the increase of Mw resulted in slightly higher work of adhesion of the nanofibers.

Development of electrosprayed mucoadhesive chitosan microparticles.

The efficacy of chitosan (CS) to be used as drug delivery carrier has previously been reported. However, limited work has been pursued to produce stable and mucoadhesive CS electrosprayed particles for oral drug delivery, which is the aim of this study. Various CS types with different molecular weight (MW), degree of deacetylation (DD), and degree of polymerization (DP) were assessed. In addition, the effect of the solvent composition was also investigated. Results showed that stable CS electrosprayed particles can be produced by dissolving 3% w/v of low MW CS in mixtures of aqueous acetic acid and ethanol (50/50% v/v). The stable CS particles displayed diameters of approximately 1 µm as determined by dynamic light scattering. The zeta potential of these particles was found to be approximately 40 mV confirming the mucoadhesion properties of these CS electrosprayed particles and its potential to be used as drug delivery carrier.