Search

Objectives: Anti-citrullinated protein/peptide antibodies (ACPA) represent an important tool for the diagnosis of rheumatoid arthritis (RA) and the presence of multiple ACPA specificities is highly correlated with the evolution towards RA. However, little is known about the association of single specificities with disease manifestations and response to therapy in established RA. The aim of this work is to evaluate in a retrospective study the clinico-serological association of ACPA detected using VCP1 and VCP2 (EBV-derived citrullinated peptides) and HCP1 and HCP2 (histone-H4-derived citrulinated peptides) in established RA., Methods: In 413 RA patients, anti-VCP1, -VCP2, -HCP1, -HCP2 were measured by ELISA. Patients were evaluated for systemic involvement, disease activity/severity, ongoing and past therapies. Data were analyzed by cluster analysis (CA) and principal component analysis (PCA)., Results: Anti-VCP1 were detected in 44% of RA patients; anti-VCP2 in 52%; anti-HCP1 in 46% and anti-HCP2 in 63%. CA and PCA independently demonstrated that ACPA levels are associated with RF positivity, and lung involvement. Subdividing patients in 5 groups according to the number of anti-peptide antibodies, mean antibody level and RF positivity, as well as the frequency of lung involvement, progressively increase in parallel with the number of ACPA specificities., Conclusions: Higher number/levels of ACPA subtypes is associated with lung involvement but not with erosive disease. Moreover, a broader ACPA repertoire may identify patients treated with biological therapy, probably affected by a more severe disease. In conclusion, ACPA typing might be relevant for a better characterization of some disease features in established RA., (Copyright © 2017 Société française de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.)

In the present work, the preparation, characterization and therapeutic potential of baicalin-loaded nanohydrogels are reported. The nanohydrogels were prepared by sonicating (S nanohydrogel) or autoclaving (A nanohydrogel) a dispersion of cholesterol-derivatized gellan in phosphate buffer. The nanohydrogel obtained by autoclave treatment showed the most promising results: smaller particles (∼362 nm vs. ∼530 nm), higher homogeneity (polydispersity index = ∼0.24 vs. ∼0.47), and lower viscosity than those obtained by sonication. In vitro studies demonstrated the ability of the nanohydrogels to favour the deposition of baicalin in the epidermis. A high biocompatibility was found for baicalin-loaded nanohydrogels, along with a great ability to counteract the toxic effect induced by hydrogen peroxide in cells, as the nanohydrogels re-established the normal conditions (∼100% viability). Further, the potential of baicalin-loaded nanohydrogels in skin wound healing was demonstrated in vivo in mice by complete skin restoration and inhibition of specific inflammatory markers (i.e., myeloperoxidase, tumor necrosis factor-α, and oedema)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Curcumin is an antimalarial compound easy to obtain and inexpensive, having shown little toxicity across a diverse population. However, the clinical use of this interesting polyphenol has been hampered by its poor oral absorption, extremely low aqueous solubility and rapid metabolism. In this study, we have used the anionic copolymer Eudragit ® S100 to assemble liposomes incorporating curcumin and containing either hyaluronan (Eudragit-hyaluronan liposomes) or the water-soluble dextrin Nutriose ® FM06 (Eudragit-nutriosomes). Upon oral administration of the rehydrated freeze-dried nanosystems administered at 25/75 mg curcumin·kg −1 ·day −1 , only Eudragit-nutriosomes improved the in vivo antimalarial activity of curcumin in a dose-dependent manner, by enhancing the survival of all Plasmodium yoelii -infected mice up to 11/11 days, as compared to 6/7 days upon administration of an equal dose of the free compound. On the other hand, animals treated with curcumin incorporated in Eudragit-hyaluronan liposomes did not live longer than the controls, a result consistent with the lower stability of this formulation after reconstitution. Polymer-lipid nanovesicles hold promise for their development into systems for the oral delivery of curcumin-based antimalarial therapies.

Background: Dysregulated production of cytokines has a critical role in systemic lupus. The aim of this work is to identify, by a comprehensive analysis of IL-1 family cytokines and receptors in serum, correlation between cytokines/receptors' levels and the clinical and serological features of the disease., Methods: A full clinical evaluation was performed in 74 patients with systemic lupus erythematosus (SLE). C3, C4, anti-dsDNA and anti-C1q antibodies were measured. Cytokines of the IL-1 family (IL-1α, IL-1β, IL-33, IL-18), soluble receptors (sIL-1R1, sIL-1R2, sIL-1R3, ST2/sIL-1R4) and antagonists (IL-1Ra, IL-18 binding protein (IL-18BP)) were measured in serum by multiarray ELISA. Free IL-18 was calculated as the amount of IL-18 not inhibited by IL-18BP. Data were analysed by non-parametric tests and by multivariate analysis, using partial least squares (PLS) models., Results: Total IL-18, IL-18BP, sIL-1R4 and IL-1Ra levels were higher in SLE vs., Controls: Total and free IL-18 and sIL-1R4 were higher in patients with active vs. inactive disease and correlated with ECLAM, anti-C1q and anti-dsDNA antibodies. sIL-1R2 was higher in patients with inactive disease, was negatively correlated with ECLAM and anti-C1q antibodies and was positively correlated with C3 levels. PLS identified sIL-1R4, sIL-1R2 and anti-dsDNA as variables distinguishing patients with active from those with inactive disease; sIL-1R4, IL-18BP and anti-dsDNA identified patients with active nephritis; sIL-1R4, C3, IL-18 and free IL-18 identified patients with haematological involvement., Conclusion: The data support the use of IL-18, sIL-1R2 and sIL-1R4 as biomarkers of disease activity and organ involvement, and suggest that failure in the inhibition of IL-1 activation may be a critical event in the active stages of SLE.

Gellan nanohydrogel and phospholipid vesicles were combined to incorporate baicalin in new self-assembling core-shell gellan-transfersomes obtained by an easy, scalable method. The vesicles were small in size (~107 nm) and monodispersed (P.I. ≤ 0.24), forming a viscous system (~24 mPa/s) as compared to transfersomes (~1.6 mPa/s), as confirmed by rheological studies. Gellan was anchored to the bilayer domains through cholesterol, and the polymer chains were distributed onto the outer surface of the bilayer, thus forming a core-shell structure, as suggested by SAXS analyses. The optimal carrier ability of core-shell gellan-transfersomes was established by the high deposition of baicalin in the skin (~11% in the whole skin), especially in the deeper tissue (~8% in the dermis). Moreover, their ability to improve baicalin efficacy in anti-inflammatory and skin repair tests was confirmed in vivo in mice, providing the complete skin restoration and inhibiting all the studied inflammatory markers., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Nutriosomes, new phospholipid nanovesicles specifically designed for intestinal protection were developed by simultaneously loading a water-soluble dextrin (Nutriose® FM06) and a natural antioxidant (curcumin). Nutriosomes were easily fabricated in a one-step, organic solvent-free procedure. The stability and delivery performances of the vesicles were improved by adding hydroxypropyl methylcellulose. All the vesicles were small in size (mean diameter ∼168 nm), negatively charged (zeta potential ∼-38 mV, irrespective of their composition), and self-assembled predominantly in unilamellar vesicles stabilized by the presence of Nutriose®, which was located in both the inter-lamellar and inter-vesicle media, as confirmed by cryo-TEM and SAXS investigation. The dextrin acted also as a cryo-protector, avoiding vesicle collapse during the lyophilization process, and as a protector against high ionic strength and pH changes encountered in the gastrointestinal environment. Thanks to the antioxidant properties of curcumin, nutriosomes provided an optimal protective effect against hydrogen peroxide-induced oxidative stress in Caco-2 cells. Moreover, these innovative vesicles showed promising efficacy in vivo, as they improved the bioavailability and the biodistribution of both curcumin and dextrin upon oral administration, which acted synergically in reducing colonic damage chemically induced in rats.

Previous work has shown that potentiation of insulin release is impaired in non-diabetic insulin resistance; we tested the hypothesis that this defect may be related to altered glucagon-like peptide-1 (GLP-1) release. On consecutive days, 82 non-diabetic individuals, classified as insulin sensitive (IS, n =41) or insulin resistant (IR, n =41) by the euglycaemic clamp, were given two sequential mixed meals with standard (75 g, LCD) or double (150 g, HCD) carbohydrate content. Plasma glucose, insulin, C-peptide, non-esterified fatty acids (NEFA) and GLP-1 concentrations were measured; β-cell function (glucose sensitivity and potentiation) was resolved by mathematical modelling. Fasting GLP-1 levels were higher in IR than IS (by 15%, P =0.006), and reciprocally related to insulin sensitivity after adjustment for sex, age, fat mass, fasting glucose or insulin concentrations. Mean postprandial GLP-1 responses were tightly correlated with fasting GLP-1, were higher for the second than the first meal, and higher in IR than IS subjects but only with LCD. In contrast, incremental GLP-1 responses were higher during (i) the second than the first meal, (ii) on HCD than LCD, and (iii) significantly smaller in IR than IS independently of meal and load. Potentiation of insulin release was markedly reduced in IR vs IS across meal and carbohydrate loading. In the whole dataset, incremental GLP-1 was directly related to potentiation, and both were inversely related to mean NEFA concentrations. We conclude that (a) raised GLP-1 tone may be inherently linked with a reduced GLP-1 response and (b) defective post-meal GLP-1 response may be one mechanism for impaired potentiation of insulin release in insulin resistance., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Glycerosomes were formulated using 1,2-dimyristoyl-sn-glycero-3-phosphatidycholine (DMPC), diclofenac sodium salt and 10, 20 or 30% glycerol in the water phase, while corresponding liposomes were prepared with the same amount of DMPC and diclofenac, without glycerol. The aim of the present work was to evaluate the effect of the used phospholipid on vesicle features and ability to favour diclofenac skin deposition by comparing these results with those found in previous works performed using hydrogenated soy phosphatidylcholine (P90H) and dipalmitoylphosphatidylcholine (DPPC). Liposomes and glycerosomes were multilamellar, liposomes being smaller (72±6nm). Interactions among glycerol, phospholipids and drug led to the formation of a non-rigid bilayer structure and a variation of the main transition temperature, which shifted to lower temperature. The addition of glycerol led to the formation of more viscous systems (from ∼2.5mPa/s for basic liposomes to ∼5mPa/s for glycerosomes), which improved spread ability of the formulations on the skin.Results obtained in vitro were promising using glycerosomes, irrespective of the amount of glycerol used: the amount of drug, which accumulated into and permeated through the different skin strata, was high and comparable with that obtained using P90H, suggesting that glycerosomes may represent an efficient carrier for both local effect or systemic absorption., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aim: Therapeutic efficacy of pulmonary diseases is often limited and drug delivery systems offer new solutions to clinical problems. Solid lipid microparticles (SLMs) are suggested as systems for the delivery of therapeutics to the lung as, because of their size, they are able to deposit into secondary bronchi., Materials & Methods: Here, we describe two novel different SLMs using chitosan and alginate such as mucoadhesive polymers and we also studied their biocompatibility and their effectiveness compared with the free drug in controlling senescence and inflammatory processes in cigarette smoke extracts., Results: Data reported show that fluticasone propionate (FP)-loaded SLMs are more effective than FP alone in controlling oxidative stress., Conclusion: The therapeutic approach using FP-loaded microparticles could be a promising strategy for the treatment of the chronic inflammatory pulmonary diseases.

Aiming at improving the nebulization performances and lung antioxidant protection of curcumin, chitosan or hyaluronan-coated liposomes were prepared and their characteristics and performances were compared with that of uncoated liposomes. Curcumin loaded liposomes displayed a diameter lower than 100nm, the coating with both polymers led to a small increase of vesicle size around 130nm and the zeta potential turned to positive values using chitosan while remained negative using hyaluronan. Chitosan allowed the formation of more lamellar and stiffer vesicles with a higher bilayer thickness (d B ∼59Ǻ) with respect to the uncoated liposomes, whereas hyaluronan allowed the interdigitation of the bilayers (d B ∼47Ǻ) due to the polymer intercalation between phospholipid head groups resulting in vesicles mainly organized in uncorrelated bilayers. Both polymer coatings, especially hyaluronan, greatly improved the stability of the vesicles, especially during the nebulization process, promoting the deposition of the phytodrug in the furthest stages of the impactor in high amount (≥50%). Polymer coated vesicles were biocompatible and improved the curcumin ability to protect A549 cells from the oxidative stress induced by hydrogen peroxide, restoring healthy conditions (cell relative metabolic activity 100%). In particular, a synergic effect of curcumin and hyaluronan was observed resulting in a proliferative effect and a subsequent further enhancement of cell relative metabolic activity up to 120%., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this study, the active components of grape pomaces were first extracted by maceration in ethanol and propylene glycol, then in extra virgin olive oil. The main components of the hydrophilic extractive solutions were flavonoids, while monounsaturated fatty acids were the most abundant constituents of the extractive oil, with high levels of oleic acid, which were identified by HPLC/DAD and GC/MS, respectively. The hydrophilic extractive solutions and the lipophilic extractive oil were used to prepare phospholipid vesicles, avoiding the energetically and economically expensive steps required to obtain solid matrixes or pure compounds. The obtained grape bioactive enriched penetration enhancer containing vesicles (PEVs) were multilamellar, around 200nm in size, and more viscous than the corresponding solutions. The antioxidant activity, evaluated by the Folin-Ciocalteu and DPPH assays, was potentiated when the extractive solutions were loaded in PEVs. Further, the grape enriched PEVs were able to ensure an optimal protection against oxidative stress in an ex vivo human erythrocytes-based model., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Gels are extensively studied in the drug delivery field because of their potential benefits in therapeutics. Depot gel systems fall in this area, and the interest in their development has been focused on long-lasting, biocompatible, and resorbable delivery devices. The present work describes a new class of hybrid gels that stem from the interaction between liposomes based on P90G phospholipid and the cholesterol derivative of the polysaccharide gellan. The mechanical properties of these gels and the delivery profiles of the anti-inflammatory model drug diclofenac embedded in such systems confirmed the suitability of these hybrid gels as a good candidate for drug depot applications., Competing Interests: The authors declare no conflict of interest.

Resveratrol and gallic acid were co-loaded in phospholipid vesicles aiming at protecting the skin from external injuries, such as oxidative stress and microbial infections. Liposomes were prepared using biocompatible phospholipids dispersed in water. To improve vesicle stability and applicability, the phospholipids and the phenols were dispersed in water/propylene glycol or water/glycerol, thus obtaining PEVs and glycerosomes, respectively. The vesicles were characterized by size, morphology, physical stability, and their therapeutic efficacy was investigated in vitro. The vesicles were spherical, unilamellar and small in size: liposomes and glycerosomes were around 70nm in diameter, while PEVs were larger (∼170nm). The presence of propylene glycol or glycerol increased the viscosity of the vesicle systems, positively affecting their stability. The ability of the vesicles to promote the accumulation of the phenols (especially gallic acid) in the skin was demonstrated, as well as their low toxicity and great ability to protect keratinocytes and fibroblasts from oxidative damage. Additionally, an improvement of the antimicrobial activity of the phenols was shown against different skin pathogens. The co-loading of resveratrol and gallic acid in modified phospholipid vesicles represents an innovative, bifunctional tool for preventing and treating skin affections., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Resveratrol and gallic acid, a lipophilic and a hydrophilic phenol, were co-loaded in innovative, biocompatible nanovesicles conceived for ensuring the protection of the skin from oxidative- and inflammatory-related affections. The basic vesicles, liposomes and glycerosomes, were produced by a simple, one-step method involving the dispersion of phospholipid and phenols in water or water/glycerol blend, respectively. Liposomes and glycerosomes were modified by the addition of poloxamer, a stabilizer and viscosity enhancer, thus obtaining viscous or semisolid dispersions of structured vesicles. The vesicles were spherical, unilamellar and small in size (~70 nm in diameter). The superior ability of the poloxamer-structured vesicles to promote the accumulation of both phenols in the skin was demonstrated, as well as their low toxicity and great ability to protect fibroblasts from chemically-induced oxidative damage. The in vivo administration of the vesicular phenols on TPA (phorbol ester)-exposed skin led to a significant reduction of oedema and leukocyte infiltration., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In the present work, silver nanoparticles were prepared using a totally green procedure combining silver nitrate and an extract of grape pomace as a green source. Additionally, nanoparticles were stabilized using phospholipid and water and/or a mixture of water and propylene glycol (PG). To the best of our knowledge, grape-silver nanoparticle stabilized liposomes or PG-liposomes were formulated, for the first time, combining the residual products of wine-made industry, silver nitrate and phospholipids, avoiding the addition of hazardous substances to human health and the environment, in an easy, scalable and reproducible method. The structure and morphology of grape-silver nanoparticle stabilized vesicles were evaluated by transmission electron microscopy (TEM), UV-vis spectroscopy and photon correlation spectroscopy. Samples were designed as possible carrier for skin protection because of their double function: the grape extract acts as antioxidant and the colloidal silver as antimicrobial agent, which might be helpful in eliminating dangerous free radicals and many pathogenic microorganisms. Obtained nanoparticles were small in size and their combination with phospholipids did not hamper the vesicle formation, which were multilamellar and sized ~100nm. TEM images shows a heterogeneous distribution of nanoparticles, which were located both in the intervesicular medium and in the vesicular structure. Further, grape-silver nanoparticles, when stabilized by liposomes, were able to inhibit the proliferation of both Staphylococcus aureus and Pseudomonas aeruginosa and provided a great protection of keratinocytes and fibroblasts against oxidative stress avoiding their damage and death., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The pomaces from red grapes were used as a source of phenolic antioxidants, which are known to have health-promoting effects. Environmentally-friendly extraction strategies were investigated to improve the rate and recovery of an extract with high phenolic content and antioxidant activity, which were evaluated by the Folin-Ciocalteu, DPPH, ABTS(+), CUPRAC and FRAP assays. The extract was incorporated in liposomes, which were stabilized by the addition of a natural polysaccharide, sodium alginate or arabic gum, widely used in pharmaceutical and food industries as thickeners and stabilizers. Results showed that the polymer-associated liposomes were approximately 300nm in size, spherical in shape, and with high entrapment efficiency. The polymers prevented vesicle degradation in the gastric environment, and played a key role in improving liposomes' performances, especially arabic gum. The polymer-associated liposomes were biocompatible and protected Caco-2 cells against oxidative stress. The achieved results suggest a potential application of the polymer-associated liposomes loaded with the grape pomace extract in the nutraceutical field., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This work aimed at finding an innovative vesicle-type formulation able to improve the bioavailability of curcumin upon oral administration. To this purpose, phospholipid, Eudragit® S100 and hyaluronan sodium salt were combined to obtain eudragit-hyaluronan immobilized vesicles using an easy and environmentally-friendly method. For the first time, the two polymers were combined in a system intended for oral delivery, to enhance curcumin stability when facing the harsh environment of the gastrointestinal tract. Four different formulations were prepared, keeping constant the amount of the phospholipid and varying the eudragit-hyaluronan ratio. The freeze-drying of the samples, performed to increase their stability, led to a reduction of vesicle size and a good homogeneity of the systems, after simple rehydration with water. X-ray diffraction study demonstrated that after the freeze-drying process, curcumin remained successfully incorporated within the vesicles. All the vesicles displayed similar features: size ranging from 220 to 287nm, spherical or oval shape, multilamellar or large unilamellar morphology with a peculiar multicompartment organization involving 1-4 smaller vesicles inside. In vitro studies demonstrated the ability of the combined polymers to protect the vesicles from the harsh conditions of the gastro-intestinal tract (i.e., ionic strength and pH variation), which was confirmed in vivo by the greater deposition of curcumin in the intestinal region, as compared to the free drug in dispersion. This enhanced accumulation of curcumin provided by the eudragit-hyaluronan immobilized vesicles, together with an increase in Caco-2 cell viability exposed to hydrogen peroxide, indicated that vesicles can ensure a local protection against oxidative stress and an increase in its intestinal absorption., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The topical efficacy of baicalin, a natural flavonoid isolated from Scutellaria baicalensis Georgi, which has several beneficial properties, such as antioxidative, antiviral, anti-inflammatory and antiproliferative, is hindered by its poor aqueous solubility and low skin permeability. Therefore, its incorporation into appropriate phospholipid vesicles could be a useful tool to improve its local activity. To this purpose, baicalin at increasing concentrations up to saturation, was incorporated in ultradeformable vesicles, which were small in size (∼67nm), monodispersed (PI<0.19) and biocompatible, regardless of the concentration of baicalin, as confirmed by in vitro studies using fibroblasts. On the other hand, transdermal flux through human epidermis was concentration dependent. The in vivo results showed the significant anti-inflammatory activity of baicalin loaded nanovesicles irrespective of the concentration used, as they were able to reduce the skin damage induced by the phorbol ester (TPA) application, even in comparison with dexamethasone, a synthetic drug with anti-inflammatory properties. Overall results indicate that ultradeformable vesicles are promising nanosystems for the improvement of cutaneous delivery of baicalin in the treatment of skin inflammation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this work, diclofenac was encapsulated, as sodium salt, in glycerosomes containing 10, 20 or 30% of glycerol in the water phase with the aim to ameliorate its topical efficacy. Taking into account previous findings, glycerosome formulation was modified, in terms of economic suitability, using a cheap and commercially available mixture of hydrogenated soy phosphatidylcholine (P90H). P90H glycerosomes were spherical and multilamellar; photon correlation spectroscopy showed that obtained vesicles were ∼131nm, slightly larger and more polydispersed than those made with dipalmitoylphosphatidylcholine (DPPC) but, surprisingly, they were able to ameliorate the local delivery of diclofenac, which was improved with respect to previous findings, in particular using glycerosomes containing high amount of glycerol (20 and 30%). Finally, this drug delivery system showed a high in vitro biocompatibility toward human keratinocytes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

[This corrects the article DOI: 10.1371/journal.pone.0157962.].

Rifampicin loaded glycerosomes, vesicles composed of phospholipids, glycerol and water, were combined with trimethyl chitosan chloride (TMC) to prepare TMC-glycerosomes or, alternatively, with sodium hyaluronate (HY) to obtain HY-glycerosomes. These new hybrid nanovesicles were tested as carriers for pulmonary delivery of rifampicin. Glycerosomes without polymers were also prepared and characterized. All vesicles were similar: they were spherical, multilamellar and able to incorporate good amount of rifampicin (EE%∼55%). The addition of the polymers to the formulations allowed an increase of mean diameter. All the glycerosomes, in particular HY-glycerosomes, were able to deliver the drug to the furthest stages of the Next Generation Impactor and the aptitude of the vesicles to be nebulized was always higher than that of drug dispersion. Rifampicin nanoincorporation in vesicles reduced the in vitro drug toxicity on A549 cells, as well as increased its efficacy against Staphylococcus aureus. Finally, the in vivo biodistribution and accumulation, evaluated after intra-tracheal administration to rats, confirmed the improvement of rifampicin accumulation in lungs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The components of pompia, a hybrid Citrus species cultivated only in Sardinia (Italy), were extracted using an environmentally-friendly method and food-grade solvents. Taking into account that only few data are available on pompia composition, the phytochemical fingerprint of its rind extract was obtained by accurate component separation and identification, combining HPLC and mass spectrometry. Different flavones such as naringin (23.77μg/mg), neoeriocitrin (46.53μg/mg) and neohesperidin (44.57μg/mg) were identified. Additionally, the antioxidant activity and phenolic content were confirmed by DPPH and Folin-Ciocalteu assays. The whole extract was incorporated in innovative phospholipid vesicles, namely glycerosomes, hyalurosomes and glycerol containing hyalurosomes, which were prepared using a high ratio of extract/phospholipid (1/3.5w/w). The in vitro biocompatibility of the nanoincorporated extract and its ability to potentiate the aptitude of the extract to counteract oxidative stress in skin cells were evaluated. The vesicles, especially glycerol containing hyalurosomes, were able to prevent oxidative damage and death of both keratinocytes and fibroblasts, promoting their viability., (Copyright © 2016 Elsevier B.V. All rights reserved.)

New biocarriers, named santosomes, were formulated using Santolina insularis essential oil and hydrogenated phosphatidylcholine. They were modified by adding propylene glycol, a hydrophylic penetration enhancer, and loaded with phycocyanin, a protein found in cyanobacteria, which possesses antioxidant and antiinflammatory properties. The essential oil was expected to modify the bilayer structure and improve the delivery and efficacy of the protein due to a synergistic effect of the phospholipid and S. insularis terpenes. Santosomes were small in size (∼118nm), unilamellar and with polyhedral shape. SAXS patterns showed that phycocyanin strongly interacted with the polar heads of the vesicle bilayer. Phycocyanin-loaded vesicles did not show any toxic effect in vitro: cell viability was ∼100% in endothelial cells and ∼120% in keratinocytes, at all the concentrations tested. In addition, phycocyanin-loaded vesicles protected the cells against free radical damage. In vivo studies were performed to evaluate the ability of santosomes to inhibit chemically-induced oedema and inflammation in mice. Results demonstrated that the application of phycocyanin-loaded santosomes produced an evident amelioration of the skin lesion, confirming their great potential for wound healing., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Allantoin is traditionally employed in the treatment of skin ulcers and hypertrophic scars. In the present work, to improve its local deposition in the skin and deeper tissues, allantoin was incorporated in conventional liposomes and in new argan oil enriched liposomes. In both cases, obtained vesicles were unilamellar, as confirmed by cryo-TEM observation, but the addition of argan oil allowed a slight increase of the mean diameter (∼130nm versus ∼85nm). The formulations, especially those containing argan oil, favoured the allantoin accumulation in the skin, in particular in the dermis (∼8.7μg/cm(2)), and its permeation through the skin (∼33μg/cm(2)). The performances of vesicles as skin delivery systems were compared with those obtained by water dispersion of allantoin and the commercial gel, Sameplast(®). Moreover, in this work, for the first time, the elastic and viscous moduli of the skin were measured, underlining the different hydrating/moisturizing effects of the formulations. The application of ARG liposomes seems to provide a softening and relaxing effect on the skin, thus facilitating the drug accumulation and passage into and trough it., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The phycobiliprotein phycocyanin, extracted from Klamath algae, possesses important biological properties but it is characterized by a low bioavailability due to its high molecular weight. To overcome the bioavailability problems, phycocyanin was successfully encapsulated, using an environmentally-friendly method, into hyalurosomes, a new kind of phospholipid vesicles immobilised with hyaluronan sodium salt by the simple addition of drug/sodium hyaluronate water dispersion to phospholipids. Liposomes were used as a comparison. Vesicles were small in size and homogeneously dispersed, being the mean size always smaller than 150 nm and PI never higher than 0.31. Liposomes were unilamellar and spherical, the addition of the polymer slightly modify the vesicular shape which remain spherical, while the addition of PEG improve the lamellarity of vesicles being multilamellar vesicles. In all cases phycocyanin was encapsulated in good amount especially using hyalurosomes and PEG hyalurosomes (65 and 61% respectively). In vitro penetration studies suggested that hyalurosomes favoured the phycocyanin deposition in the deeper skin layers probably thanks to their peculiar hyaluronan-phospholipid structure. Moreover, hyalurosomes were highly biocompatible and improved phycocyanin antioxidant activity on stressed human keratinocytes respect to the drug solution.

Context: Renal disease in type 2 diabetes mellitus (T2DM) is associated with excess morbidity/mortality. Although estimated glomerular filtration rate (eGFR) and albuminuria are routine for assessing renal impairment, novel biomarkers could improve risk stratification and prediction., Objective: To identify specific biomarkers of progression of renal dysfunction., Design: Prospective observational., Setting: Academic diabetes clinics., Patients: A total of 286 T2DM patients (age, 62 ± 8 y; glycosylated hemoglobin, 7.2 ± 0.9%; eGFR, 85 ± 20 mL · min(-1) · 1.73 m(2))., Interventions: None., Main Outcome Measures: Progression of eGFR and albuminuria., Results: We performed screening metabolomics in serum and urine samples by gas chromatography/mass spectroscopy (MS) and ultra-high performance liquid chromatography/MS/MS. Biomarker identification was performed by random forest using an eGFR cutoff of < 60 mL · min(-1) · 1.73 m(2) or an albumin/creatinine ratio (ACR) cutoff ≥ 30 mg/g as response variables. At follow-up, eGFR had declined by 16 [9] (median [interquartile ratio]) mL · min(-1) · 1.73 m(2), and ACR had increased by 41 [135] mg/g in patients in the respective top quartile of changes from baseline. Clinical parameters (gender, age, fasting glucose, and baseline eGFR) predicted outcome, with receiver operator characteristics curve (ROC) = 0.671. The five serum metabolites best correlated with either eGFR < 60 or ACR ≥ 30 at baseline were tested for their ability to improve clinical prediction. The sum of C-glycosyl tryptophan, pseudouridine, and N-acetylthreonine (MetIndex) raised the ROC to 0.739 (P < .0001). eGFR decline was predicted by the top MetIndex quartile (odds ratio = 5.48 [95% confidence interval, 2.23-14.47]). MetIndex also predicted an ACR increase with an odds ratio of 2.82 [1.20-7.03] and a ROC of 0.750. Top urine metabolites did not add significant predictivity., Conclusions: A limited number of circulating intermediates of amino acid and nucleotide pathways carry clinically significant predictivity for deterioration of renal function in well-controlled T2DM.

Liquorice extract, obtained by percolation in ethanol of Glycyrrhiza glabra L. roots, was incorporated in liposomes and hyalurosomes, new phospholipid-sodium hyaluronate vesicles, and their protective effect against oxidative stress skin damages was probed. As a comparison, raw glycyrrhizin was also tested. All the vesicles were small in size (≤ 100 nm), with a highly negative zeta potential ensuring long-term stability, and able to incorporate a high amount of the extract. In vitro tests showed that the liquorice extract loaded in vesicles was able to scavenge DPPH free radical (80% inhibition) and to protect 3T3 fibroblasts against H2O2-induced oxidative stress, restoring the normal conditions. By contrast, glycyrrhizin showed poor antioxidant activity, and was not able to efficiently counteract the oxidative effect of H2O2. In addition, the incorporation of the liquorice extract into the vesicular systems promoted the proliferation and migration of 3T3 fibroblasts, favouring the closure of the scratched area. In vivo anti-inflammatory tests on mice confirmed the ability of the proposed nanosystems to improve the local efficacy of the extract, favouring the re-epitelization process., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the present work new highly biocompatible nanovesicles were developed using polyanion sodium hyaluronate to form polymer immobilized vesicles, so called hyalurosomes. Curcumin, at high concentration was loaded into hyalurosomes and physico-chemical properties and in vitro/in vivo performances of the formulations were compared to those of liposomes having the same lipid and drug content. Vesicles were prepared by direct addition of dispersion containing the polysaccharide sodium hyaluronate and the polyphenol curcumin to a commercial mixture of soy phospholipids, thus avoiding the use of organic solvents. An extensive study was carried out on the physico-chemical features and properties of curcumin-loaded hyalurosomes and liposomes. Cryogenic transmission electron microscopy and small-angle X-ray scattering showed that vesicles were spherical, uni- or oligolamellar and small in size (112-220 nm). The in vitro percutaneous curcumin delivery studies on intact skin showed an improved ability of hyalurosomes to favour a fast drug deposition in the whole skin. Hyalurosomes as well as liposomes were biocompatible, protected in vitro human keratinocytes from oxidative stress damages and promoted tissue remodelling through cellular proliferation and migration. Moreover, in vivo tests underlined a good effectiveness of curcumin-loaded hyalurosomes to counteract 12-O-tetradecanoilphorbol (TPA)-produced inflammation and injuries, diminishing oedema formation, myeloperoxydase activity and providing an extensive skin reepithelization. Thanks to the one-step and environmentally-friendly preparation method, component biocompatibility and safety, good in vitro and in vivo performances, the hyalurosomes appear as promising nanocarriers for cosmetic and pharmaceutical applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The aim of this work was to formulate Santolina insularis essential oil-loaded nanocarriers, namely Penetration Enhancer containing Vesicles (PEVs), evaluate the physico-chemical features and stability, and gain insights into their ability to deliver the oil to the skin. S. insularis essential oil was obtained by steam distillation, and was predominantly composed of terpenes, the most abundant being β-phellandrene (22.6%), myrcene (11.4%) and curcumenes (12.1%). Vesicles were prepared using phosphatidylcholine, and ethylene or propylene glycol were added to the water phase (10% (v/v)) to improve vesicle performances as delivery systems. Vesicles were deeply characterized by light scattering, cryogenic transmission electron microscopy and small/wide-angle X-ray scattering, the results showing polyhedral, faceted, unilamellar vesicles of ∼115 nm in size. The presence of the glycols improved vesicle stability under accelerated ageing conditions, without changes in size or migration phenomena (e.g. sedimentation and creaming). Confocal laser scanning microscopy images of pig skin treated with S. insularis formulations displayed a penetration ability of PEVs greater than that of control liposomes. Moreover, all formulations showed a marked in vitro biocompatibility in human keratinocytes. These findings suggest that the nanoformulation may be of value in enhancing the delivery of S. insularis essential oil to the skin, where it can exert its biological activities., (Copyright © 2015 Elsevier B.V. All rights reserved.)

High-carbohydrate diets have been associated with β-cell strain, dyslipidemia, and endothelial dysfunction. We examined how β-cell and endothelial function adapt to carbohydrate overloading and the influence of insulin resistance. On sequential days in randomized order, nondiabetic subjects (classified as insulin-sensitive [IS] [n = 64] or insulin-resistant [IR] [n = 79] by euglycemic clamp) received four mixed meals over 14 h with either standard (300 kcal) or double carbohydrate content. β-Cell function was reconstructed by mathematical modeling; brachial artery flow-mediated dilation (FMD) was measured before and after each meal. Compared with IS, IR subjects showed higher glycemia and insulin hypersecretion due to greater β-cell glucose and rate sensitivity; potentiation of insulin secretion, however, was impaired. Circulating free fatty acids (FFAs) were less suppressed in IR than IS subjects. Baseline FMD was reduced in IR, and postprandial FMD attenuation occurred after each meal, particularly with high carbohydrate, similarly in IR and IS. Throughout the two study days, higher FFA levels were significantly associated with lower (incretin-induced) potentiation and impaired FMD. In nondiabetic individuals, enhanced glucose sensitivity and potentiation upregulate the insulin secretory response to carbohydrate overloading. With insulin resistance, this adaptation is impaired. Defective suppression of endogenous FFA is one common link between impaired potentiation and vascular endothelial dysfunction., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Plasma glucose thresholds for diagnosis of type 2 diabetes are currently based on outcome data (risk of retinopathy), an inherently ill-conditioned approach. A radically different approach is to consider the mechanisms that control plasma glucose, rather than its relation to an outcome. We developed a constraint optimization algorithm to find the minimal glucose levels associated with the maximized combination of insulin sensitivity and β-cell function, the two main mechanisms of glucose homeostasis. We used a training cohort of 1,474 subjects (22% prediabetic, 7.7% diabetic) in whom insulin sensitivity was measured by the clamp technique and β-cell function was determined by mathematical modeling of an oral glucose tolerance test. Optimized fasting glucose levels were ≤ 87 and ≤ 89 mg/dl in ≤ 45-yr-old women and men, respectively, and ≤ 92 and ≤ 95 mg/dl in >45-yr-old women and men, respectively; the corresponding optimized 2-h glucose levels were ≤ 96, ≤ 98, ≤ 103, and ≤ 105 mg/dl. These thresholds were validated in three prospective cohorts of nondiabetic subjects (Relationship Between Insulin Sensitivity and Cardiovascular Disease Study, Botnia Study, and Mexico City Diabetes Study) with baseline and follow-up oral glucose tolerance tests. Of 5,593 participants, 452 progressed to diabetes. Similarly, in the three cohorts, subjects with glucose levels above the estimated thresholds had an odds ratio of 3.74 (95% confidence interval = 2.64-5.48) of progressing, substantially higher than the risk carried by baseline conventionally defined prediabetes [odds ratio = 2.32 (95% confidence interval = 1.91-2.81)]. The concept that optimization of glucose concentrations by direct measures of insulin sensitivity and β-cell function identifies gender- and age-specific thresholds that bear on disease progression is proven in a physiologically sound, quantifiable manner., (Copyright © 2015 the American Physiological Society.)

In this study, the effects of ethanol and/or diclofenac on vesicle bilayer structure have been studied. Liposomes with hydrogenated soy phosphatidylcholine, cholesterol and two different concentrations of diclofenac sodium (5 and 10 mg/ml) were obtained. In addition, ethanol was mixed in the water phase at different concentrations (5, 10 and 20 % v/v) to obtain ethosomes. To characterize vesicles, rehological analysis were carried out to investigate the intervesicle interactions, while bilayer structure was evaluated by small- and wide-angle X-ray scattering. Finally, the ethanol and/or diclofenac concentration-dependent ability to improve diclofenac skin delivery was evaluated in vitro. The addition of 20 % ethanol and/or diclofenac led to solid-like ethosome dispersion due to the formation of a new intervesicle structure, as previously found in transcutol containing vesicle dispersions. However, when using 5-10 % of ethanol the induction to form vesicle interconnections was less evident but the simultaneous presence of the drug at the highest concentration facilitated this phenomenon. Ethosomes containing the highest amount of both, drug (10 mg/ml) and ethanol (20 % v/v), improved the drug deposition in the skin strata and in the receptor fluid up to 1.5-fold, relative to liposomes. Moreover this solid-like formulation can easily overcome drawbacks of traditional liquid liposome formulations which undergo a substantial loss at the application site.

The aim of the present study was to elucidate the polyphenolic composition of Fraxinus angustifolia leaf and bark extracts, and to evaluate their efficacy in wound healing. Quercetin, catechin, rutin and tannic acid were identified as the main components of the extracts. In order to improve their skin bioavailability, the polyphenolic phytocomplexes were incorporated in different nanovesicles, namely ethosomes and phospholipid vesicles containing Transcutol(®) P (Trc) or ethylene glycol (EG). The latter had never been used before as a component of phospholipid vesicles, and it was found to play a key role in improving extract efficacy in wound healing. Results of cryogenic transmission electron microscopy (cryo-TEM), Photon Correlation Spectroscopy (PCS) and Small-Angle X-ray Scattering (SAXS) showed that ethosomes and EG-PEVs were small, monodispersed, unilamellar vesicles, while Trc-PEVs were larger, less homogeneously dispersed and multilamellar, with a large bilayer thickness. Free extracts did not show relevant ability to protect in vitro human keratinocytes from H2O2 damages, while when entrapped in nanovesicles, they significantly inhibited H2O2 stress damages, probably related to a higher level of cell uptake. On the other hand, in vivo results showed that the highest antioxidant and anti-inflammatory effects were provided by the phytocomplexes in EG-PEVs, which favoured wound healing. Moreover, non-entrapped F. angustifolia extracts showed a marginal effect, comparable to that of free quercetin dispersion (control). In conclusion, our results depict that these extracts may find potential applications in biomedicine., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Unlabelled: This work describes innovative niosomes, composed of diolein alone or in association with the hydrophilic penetration enhancer Labrasol(®), as carriers for cutaneous drug delivery. The model drug was tretinoin and conventional, and Labrasol(®) containing liposomes was used as controls to evaluate the influence of vesicle composition and the role of Labrasol(®) on vesicle physico-chemical properties and performance as skin delivery system. Vesicles, prepared by the thin film hydration technique, were characterized in terms of size distribution, morphology, zeta potential, structure, incorporation efficiency, and rheological properties. The influence of carrier composition on tretinoin delivery to human skin was evaluated by in vitro percutaneous experiments, while formulation distribution on human skin and cellular uptake in human keratinocytes were studied using confocal laser scanning microscopy., Result: showed that tretinoin loaded diolein-niosomes formed unilamellar vesicles very similar in physico-chemical properties to liposomes. The role of Labrasol(®) was similar in niosomes and liposomes. Its addition affected vesicle structure and size, by formation of an interdigitate bilayer with higher curvature and larger vesicle size, and rheological properties. Indeed, the presence of Labrasol(®) allowed both niosomes and liposomes to shift from Newtonian to pseudo-plastic behavior. Confocal laser microscopy highlighted an important contemporaneous deposition of hydrophilic and lipophilic vesicle components in stratum corneum and a high vesicle affinity for skin appendages when Labrasol(®) was added to the diolein-niosomes. Moreover, all samples were internalized in human keratinocytes in vitro., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Quercetin was incorporated in glycerosomes, new phospholipid-glycerol vesicles, and their protective effect against oxidative stress skin damages was extensively evaluated. In particular, the concentration-dependent effect of glycerol (from 10 to 50%) on vesicle suitability as cutaneous carriers of quercetin was carefully assessed. All vesicles were unilamellar and small in size (∼80-110 nm), as confirmed by cryo-TEM observation, with a drug incorporation efficiency ranging between 81 and 91%. SAXS studies, performed to investigate the bilayer arrangement, indicated a strong, dose-dependent interaction of glycerol with the polar portions of the phospholipid molecules, while quercetin did not significantly change the bilayer packing. In vitro studies on newborn pig skin underlined the concentration-dependent ability of glycerosomes to promote quercetin accumulation in the different layers, also confirmed by confocal microscopic observation of skin treated with fluorescent vesicles. Quercetin incorporated into liposomal and glycerosomal nanoformulations showed a strong ability to scavenge free radicals (DPPH test) and protect human keratinocytes in vitro against hydrogen peroxide damage. Moreover, quercetin-loaded vesicles were avidly taken up by keratinocytes in vitro. Overall, results indicate 40 and 50% glycerosomes as promising nanosystems for the improvement of cutaneous quercetin delivery and keratinocyte protection against oxidative stress damage., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A polyelectrolyte complex based on chitosan and carrageenan was used to coat rifampicin-loaded vesicles and obtain a dry powder for inhalation by spray-drying. The polymer complexation on vesicle surface stabilized them and improved their adhesion on airways and epithelia cells. Uncoated liposomes were small in size, negatively charged and able to incorporate large amounts of rifampicin (70%). Coated vesicles were still able to load adequate amounts of drug (∼70%) but the coating process produced larger particles (1 μm) that were positively charged and with a spherical shape. Aerosol performances, evaluated using the next-generation impactor, showed that coated vesicles reached the 50% of fine particle fraction and the smallest mass median aerodynamic diameter (2 μm). Rifampicin-loaded uncoated and coated vesicles slowly reduced the A549 cell viability over a 48-h incubation time. Moreover, in vitro coated formulations had a strong ability to be easily internalized and to greatly prolong the residence time of their components in A549 cells compared to uncoated liposomes that were rapidly internalized and just as quickly removed., (Copyright © 2014. Published by Elsevier B.V.)

There are several pieces of evidence indicating that Mycobacterium avium subspecies paratuberculosis (MAP) infection is linked to type 1 diabetes (T1D) in Sardinian patients. An association between MAP and T1D was recently observed in an Italian cohort of pediatric T1D individuals, characterized by a different genetic background. It is interesting to confirm the prevalence of anti-MAP antibodies (Abs) in another pediatric population from continental Italy, looking at several markers of MAP presence. New-onset T1D children, compared to age-matched healthy controls (HCs), were tested by indirect enzyme-linked immunosorbent assay for the presence of Abs toward the immunodominant MAP3865c/ZnT8 homologues epitopes, the recently identified C-terminal MAP3865c281-287 epitope and MAP-specific protein MptD. Abs against MAP and ZnT8 epitopes were more prevalent in the sera of new-onset T1D children compared to HCs. These findings support the view that MAP3865c/ZnT8 cross-reactivity is involved in the pathogenesis of T1D, and addition of Abs against these peptides to the panel of existing T1D biomarkers should be considered. It is important now to investigate the timing of MAP infection during prospective follow-up in at-risk children to elucidate whether Ab-titers against these MAP/ZnT8 epitopes are present before T1D onset and if so if they wane after diagnosis.

A self-assembled hybrid phospholipid vesicular system containing various penetration enhancers - ethanol, Transcutol and propylenglycol - was prepared and characterized. The effects of the different alcohol or polyalcohols structure and their concentration on the features of the assembled vesicles were evaluated using a combination of different techniques, including cryo-transmission electron microscopy, laser light scattering, differential scanning calorimetry, small- and wide-angle X-ray scattering and rheological analysis. These techniques allow explaining the structural rearrangements of the bilayer assembly due to the alcohol or polyalcohol addition. X-ray scattering studies showed that such addition at the highest concentration (20%) allowed structure modification to oligolamellar vesicles and a bilayer transition to interdigitated phase. Rheological studies confirmed the importance of alcohol or polyalcohol in the structuring dispersions probably due to a partial tilting of phosphatidylcholine acyl chains forming interdigitated and interconnected bilayer vesicles., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim of the current study was to improve the knowledge of drug-glycol-phospholipid-interactions and their effects in lamellar vesicle suitability as drug delivery systems. Liposomes were prepared using hydrogenated soy phosphatidylcholine (P90H, 60 mg/ml) and diclofenac sodium salt at two concentrations (5-10 mg/ml). To obtain innovative vesicles two permeation enhancers with glycol group, diethyleneglycol monoethyl ether and propylene glycol, were added to the water phase at different ratios (5%, 10%, and 20%). Vesicle organization was deeply investigated by physico-chemical characterization, including differential scanning calorimetry and small-angle diffraction signal analysis while macroscopic structure behavior was evaluated by rheological studies. Results evidenced that the presence of the penetration enhancer and diclofenac sodium salt led to structural rearrangements within and among vesicles forming a tridimensional and complex architecture in which vesicles were closely packed and interconnected. This new design allowed a change in the physical state of dispersions that became highly viscous liquid or soft-solid-like, thus forming an ideal system for topical application able of both adhering to the skin and delivering the drug., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Concentrated and interconnected penetration enhancer containing vesicles (PEVs) are proposed as carriers for dermal delivery of diclofenac. PEVs were prepared by using a commercial phosphatidylcholine mixture (180 mg/m) and transcutol in different amounts. Conventional liposomes were also prepared and tested as control. All vesicles showed a mean size ranging from 75 to 253 nm with fairly narrow size distribution, negative zeta potential value, and drug loading capacity between 48 and 70%. SWAXS studies showed that composition affected vesicle structure and morphology: 10 and 30% transcutol PEVs were unilamellar while liposomes and 20% transcutol PEVs were multilamellar. Rheological studies demonstrated that control liposomes and 10 and 30% transcutol containing PEVs behaved as Newtonian fluids while 20% transcutol containing PEVs showed a plastic behavior. Ex vivo (trans)dermal delivery experiments showed an improved skin deposition of diclofenac when PEVs were used. Vesicle toxicity and uptake of fibroblasts, target of inflammation treatment, were evaluated by MTT test and fluorescence microscopy. Control liposomes and PEVs were both able to interact and being internalized by the 3T3 fibroblasts at all time exposure tested. Furthermore, PEVs showed to be able to reduce the in vitro drug toxicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This work describes glycerosomes, vesicles composed of phospholipids, glycerol, and water, as novel vesicular carriers for (trans)dermal drug delivery. In this work, glycerosomes were prepared by hydrating dipalmitoylglycerophosphatidylcholine-cholesterol films with glycerol aqueous solutions (10-30%, v/v). The model drug was diclofenac sodium salt and conventional liposomes were used as control. Prepared formulations were characterized in terms of size distribution, morphology, zeta potential, and vesicle deformability. Glycerosomes and liposomes were oligo/multilamellar vesicles, spherical in shape with a mean diameter ranging between 81 and 97 nm and a fairly narrow distribution (P.I.=0.14-0.19), negative zeta potential values (from -35 to -48) and drug loading capacity between 64 and 73%. Deformability index of both conventional liposomes and glycerosomes showed that glycerol is able to act as edge activator for dipalmitoylglycerophosphatidylcholine bilayers when used in concentration higher than 10%. DSC studies suggested that glycerosomes are in a more fluid state than conventional liposomes. In vitro transdermal delivery experiments showed an improved skin deposition and permeation of diclofenac when 20 and 30% glycerosomes were used. MTT test demonstrated that glycerosomes were able to reduce the in vitro drug toxicity versus keratinocytes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives: In this work, a new polymeric microparticle system based on gelatin covalently bound to isoniazid (ISN) and containing rifampicin (RFP) was prepared by spray-drying technique. Microparticle aptitude to nebulisation and their capability of interacting with A549, alveolar basal epithelial cells, were evaluated in vitro., Methods: Microparticles were obtained by spray drying, and their morphology, size, zeta potential, thermotropic behaviour and nebulisation ability were evaluated., Key Findings: Microparticles were positively charged with a mean size of 4.88 ± 0.3 μm. Microspheres were able to incorporate both RFP and ISN: encapsulation efficiency was 51 ± 6% and 22 ± 1%, respectively. X-ray diffraction study showed a new extensive and flattened diffraction peak providing evidence that the drugs were dispersed into the microparticles. Differential scanning calorimetry analysis confirmed effective interactions between gelatin and drug molecules by the presence of new transition peaks. Fifty-nine per cent of used microparticles were aerosolised. In-vitro toxicity studies on A549 alveolar basal epithelial cells showed that microparticles decreased cytotoxicity in comparison with the RFP solution. Laser scanning confocal microscopy observation confirmed that fluorescent probes delivered by microparticles are efficiently internalised in A549 cells., Conclusions: Overall, microparticles based on gelatin covalently bound to ISN and containing RFP showed a promising behaviour for pulmonary drug delivery., (© 2013 Royal Pharmaceutical Society.)

The purpose of this study was to develop a new delivery system capable of improving bioavailability and controlling release of hydrophilic drugs. Metformin-loaded liposomes were prepared and to improve their stability surface was coated with chitosan cross-linked with the biocompatible β-glycerolphosphate. X-ray diffraction, differential scanning calorimetry, as well as rheological analysis were performed to investigate interactions between chitosan and β-glycerolphosphate molecules. The entrapment of liposomes into the chitosan-β-glycerolphosphate network was assessed by scanning electron microscopy and transmission electron microscopy. Swelling and mucoadhesive properties as well as drug release were evaluated in vitro while the drug oral bioavailability was evaluated in vivo on Wistar rats. Results clearly showed that, compared to control, the proposed microcomplexes led to a 2.5-fold increase of metformin T(max) with a 40% augmentation of the AUC/D value.

A novel cationic liposome nanocarrier, having interesting performance in topical drug delivery, is here presented and evaluated for its features. Two penetration enhancers, namely monoolein and lauroylcholine chloride, are combined to rapidly formulate (15 min) a cationic liposome nanostructure endowed of excellent stability (>6 months) and skin penetration ability, along with low short-term cytotoxicity, as evaluated via the MTT test. Cytotoxicity tests and lipid droplet analysis give a strong indication that monoolein and lauroylcholine synergistically endanger long-term cells viability. The physicochemical features, investigated through SAXS, DLS, and cryo-TEM techniques, reveal that the nanostructure is retained after loading with diclofenac in its acid (hydrophobic) form. The drug release performances are studied using intact newborn pig skin. Analysis of the different skin strata proves that the drug mainly accumulates into the viable epidermis with almost no deposition into the derma. Indeed, the flux of the drug across the skin is exceptionally low, with only 1% release after 24 h. These results validate the use of this novel formulation for topical drug release when the delivery to the systemic circulation should be avoided., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The effects of lipid concentration and composition on the physicochemical properties, aerosol performance and in vitro toxicity activity of several rifampicin-loaded liposomes were investigated. To this purpose, six liposome formulations containing different amounts of soy phosphatidylcholine and hydrogenated soy phosphatidylcholine, with and without cholesterol and oleic acid, were prepared and fully characterized. Uni- or oligo-lamellar, small (~100 nm), negatively charged (~60 mV) vesicles were obtained. Lipid composition affected aerosol delivery features of liposomal rifampicin; in particular, the highest phospholipid concentration led to a better packing of the vesicular bilayers with a consequent higher nebulization stability. The retention of drug in nebulized vesicles (NER%) was higher for oleic acid containing vesicles (55% ± 1.4%) than for the other samples (~47%). A549 cells were used to evaluate intracellular drug uptake and in vitro toxicity activity of rifampicin-loaded liposomes in comparison with the free drug. Cell toxicity was more evident when oleic acid containing liposomes were used.

In cancer patients mild-moderate non-chemotherapy-induced iron deficiency anemia (IDA) is usually treated with oral iron salts, mostly ferrous sulfate. In this study, we compare efficacy and toxicity of oral ferrous bisglycinate chelate and ferrous sulfate in cancer patients with mild IDA. Twenty-four patients operated on for solid tumors (10 breast, 12 colorectal, 2 gastric), aged 61±10 years (range 45-75), with non-chemotherapy-induced hemoglobin (Hb) values between 10 and 12 g/dL and ferritin lower than 30 ng/mL were randomized to receive oral ferrous bisglycinate chelate, 28 mg per day for 20 days, and then 14 mg per day for 40 days (12 patients) (A group) or oral ferrous sulphate, 105 mg per day for 60 days (12 patients) (B group). Values of hemoglobin and ferritin obtained at diagnosis, 1 and 2 months from the beginning of treatment were compared. Adverse events (AEs) related to the two treatments were recorded. In the 12 patients treated with ferrous bisglycinate chelate, basal hemoglobin and ferritin values (mean±SD) were 11.6±0.8 g/dL and 16.1±8.0 ng/mL. After 2 months of treatment, they were 13.0±1.4 g/dL and 33.8±22.0 ng/mL, respectively (P=0.0003 and P=0.020). In the group treated with ferrous sulphate, hemoglobin and ferritin mean values were 11.3±0.6 g/dL and 19.0±6.4 ng/mL basally, and 12.7±0.70 g/dL and 40.8±28.1 ng/mL (P<0.0001 and P=0.017) after 2 months of treatment. AEs occurred in six cases. In all these six cases, two (17%) treated with ferrous bisglycinate chelate and four (33%) with ferrous sulphate, toxicity was grade 1. In conclusion, these data suggest that ferrous bisglycinate chelate has similar efficacy and likely lower GI toxicity than ferrous sulphate given at the conventional dose of 105 mg per day for the same time., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

The aim of this work was to develop new microparticles for drug delivery to lungs by coating liposomes with chitosan (CH)-xanthan gum (XG) polyelectrolyte complexes to obtain chitosomes. To this purpose, two groups of liposomes were prepared using a mixture of soy phosphatidylcholine and hydrogenated soy phosphatidylcholine in two different concentrations to evaluate their capability to entrap appropriate amounts of the model drug rifampicin. The obtained vesicles were then coated with different CH-XG weight ratios and liposomes and chitosomes were characterized in terms of morphology, size, size distribution, zeta potential, drug entrapment, and rheological properties. The efficiency of chitosomes and liposomes during nebulization was also studied. Results of this study indicated that nebulization and rheological properties of chitosomes are affected by the CH-XG weight ratio. In particular, CH-XG 1:0.5 (w/w) coating was able to greatly improve drug total mass output and drug deposition in the lower stages of the impinger., (Copyright © 2011 Wiley Periodicals, Inc.)

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants. Recent studies have linked MAP to type 1 diabetes (T1D) in the Sardinian population. The aim of this study was to investigate the prevalence of MAP infection in a T1D cohort from continental Italy compared with healthy control subjects. 247 T1D subjects and 110 healthy controls were tested for the presence of MAP. MAP DNA was detected using IS900-specific polymerase chain reaction (PCR). The presence of antibodies towards a MAP antigen, heparin binding hemoagglutinin (HBHA), was detected by ELISA. We demonstrated a higher MAP DNA prevalence in plasma samples from T1D patients and a stronger immune response towards MAP HBHA, compared with healthy control subjects. Moreover, in the recent onset patients, we observed an association between anti-MAP antibodies and HLA DQ2 (DQA1 0201/DQB1 0202). These findings taken together support the hypothesis of MAP as an environmental risk factor for the development of T1D in genetically predisposed subjects, probably involving a mechanism of molecular mimicry between MAP antigens and pancreatic islet β-cells.

In this paper, orally disintegrating tablets (ODT) were prepared using nanocrystal formulations in order to optimise dissolution properties of lipophilic, poorly soluble drug piroxicam (PRX). Different nanocrystal formulations were prepared using a high pressure homogenisation technique and poloxamer 188 as stabiliser. Characterisation of PRX nanocrystal ODT was carried out by infrared spectroscopy (FTIR), X-ray powder diffractometry (XRPD), differential scanning calorimetry and photon correlation spectroscopy. Dissolution study of PRX ODT was performed in distilled water (pH 5.5) and was compared to that of PRX coarse suspension ODT, PRX/poloxamer 188 physical mixture and bulk PRX samples. The XRPD and FTIR studies demonstrated that the homogenisation process led to a polymorphic transition from form I (bulk commercial PRX) to form III and monohydrate form of the nanocrystals. All ODT formulations prepared using PRX nanosuspensions showed a higher PRX dissolution rate compared with the ODT prepared with the coarse PRX. Since the solubility of the different PRX polymorphic forms increased only slightly from bulk PRX (form I) to monohydrate, form II and form III, we can conclude that the improvement in PRX dissolution rate is mainly caused by the increased surface-to-volume ratio due to the submicron dimension of the drug particles., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In type 1 diabetes mellitus (T1D) p53 pathways are up-regulated and there is an increased susceptibility to apoptosis. The hypothesis is that p53 codon 72 polymorphism could be associated with T1D. A total of 286 children with T1D and a control sample of 730 subjects were studied. p53 codon 72 polymorphism was analysed by polymerase chain reaction. A large increase of p53 *Arg/*Arg was observed in T1D patients with age at onset < 6 years. A strong linear correlation between *Arg/*Arg genotype and age at onset was observed in females. The involvement of the *Arg/*Arg genotype in apoptosis suggests that during the autoimmune process leading to T1D, genetic factors that favor apoptosis may contribute to the onset of overt disease.