Impact of molecular weight on the formation of electrosprayed chitosan microcapsules as delivery vehicles for bioactive compounds

The molecular weight of chitosan is one of its most determinant characteristics, which affects its processability and its performance as a biomaterial. However, information about the effect of this parameter on the formation of electrosprayed chitosan microcapsules is scarce. In this work, the impact of chitosan molecular weight on its electrosprayability was studied and correlated with its effect on the viscosity, surface tension and electrical conductivity of solutions. A Discriminant Function Analysis revealed that the morphology of the electrosprayed chitosan materials could be correctly predicted using these three parameters for almost 85% of the samples. The suitability of using electrosprayed chitosan capsules as carriers for bioactive agents was also assessed by loading them with a model active compound, (−)-epigallocatechin gallate (EGCG). This encapsulation, with an estimated efficiency of around 80% in terms of preserved antioxidant activity, showed the potential to prolong the antiviral activity of EGCG against murine norovirus via gradual bioactive release combined with its protection against degradation in simulated physiological conditions.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. Gloria Sanchez was supported by the “Ramón y Cajal” Young Investigator Program. The authors would like to thank the Spanish MINECO project AGL2015-63855-C2-1 and INIA grant RTA2014-00024-C04-03 for financial support.Peer reviewe

Protective Effects of Bovine Serum Albumin on Superparamagnetic Iron Oxide Nanoparticles Evaluated in the Nematode Caenorhabditis elegans

Nanomaterials give rise to unique biological reactivity that needs to be thoroughly investigated. The quest for enhanced magnetic nanomaterials of different shapes, magnetic properties, or surface coatings continues for applications in drug delivery, targeting therapies, biosensing, and magnetic separation. In this context, the use of simple in vivo models, such as Caenorhabditis elegans, to biologically evaluate nanoparticles is currently in increasing demand as it offers low-cost and information-rich experiments. In this work, we evaluated how surface modification (citrate- and protein-coated) of superparamagnetic iron oxide nanoparticles (C-SPIONs and BSA-SPIONs, respectively) induces changes in their toxicological profile and biodistribution using the animal model C. elegans and combining techniques from materials science and biochemistry. The acute toxicity and nanoparticle distribution were assessed in two populations of worms (adults and larvae) treated with both types of SPIONs. After 24 h treatment, nanoparticles were localized in the alimentary system of C. elegans; acute toxicity was stronger in adults and larvae exposed to C-SPIONs rather than BSA-SPIONs. Adult uptake was similar for both SPION types, whereas uptake in larvae was dependent on the surface coating, being higher for BSA-SPIONs. Nanoparticle size was evaluated upon excretion, and a slight size decrease was found. Interestingly, all results indicate the protective effects of the BSA to prevent degradation of the nanoparticles and decrease acute toxicity to the worms, especially at high concentrations. We argue that this relevant information on the chemistry and toxicity of SPIONs in vivo could not be gathered using more classical in vitro approaches such as cell culture assays, thus endorsing the potential of C. elegans to assess nanomaterials at early stages of their synthetic formulations.C. elegans N2 and E. coli OP50 were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). The research leading to these results has received funding from the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program (FP7/2007-2013) under REA grant agreement nº 303630 and cofounded by the European Social Fund. Authors acknowledge the funding from Spanish Ministry of Economy MAT 2012-35324 and FEDER funds, the Generalitat de Catalunya 2014SGR213, the COST Action MP1202, Ramon y Cajal grant RYC-2010-06082 (AL), China Scholarship Council fellowship (SMY, 201206150053), and FPU fellowship FPU12/05549 (LGM).Peer Reviewe

Optimization of electrospraying conditions for the microencapsulation of probiotics and evaluation of their resistance during storage and in-vitro digestion

Electrospraying has recently emerged as a novel microencapsulation technique with potential for the protection of probiotics. However, research efforts are still needed to minimize the viability loss observed during the processing of sensitive strains, and to maximize productivity. The aim of the present work was the optimization of the electrospraying conditions for the microencapsulation of a model probiotic microorganism, Lactobacillus plantarum, within a whey protein concentrate matrix. In a pre-optimization step, the convenience of encapsulating fresh culture instead of freeze-dried bacteria was established. Additionally, a surface response methodology was used to study the effect of the applied voltage, surfactant concentration, and addition of a prebiotic to the formulation on cell viability and productivity. Viability losses lower than 1 log10 CFU were achieved and the bacterial counts of the final products exceeded 8.5 log10 CFU/g. The protection ability of the developed structures during storage and in-vitro digestion was also evaluated.Laura G. Gómez-Mascaraque is recipient of a predoctoral contract from the Spanish Ministry of Economy and Competitiveness (MINECO), Call 2013. Russell Cruz Morfin received a scholarship from the Mexican National Council for Science and Technology (CONACYT), Call 2014. Gloria Sánchez is supported by the “Ramón y Cajal” Young Investigator program of the MINECO. This work was financially supported by the Spanish MINECO project AGL2012-30647 and by the CSIC project 201470I002.Peer reviewe

Evaluating inorganic nanoparticles in the living organism Caenorhabditis elegans /

BibliografiaPremi Extraordinari de Doctorat concedit pels programes de doctorat de la UAB per curs acadèmic 2016-2017En aquesta tesi hem utilitzat el model animal Caenorhabditis elegans (C. elegans) com a sistema biològic viu per avaluar nanopartícules (NPs) per aplicacions biomèdiques. En concret, hem avaluat el comportament de dos tipus de partícules inorgàniques amb diferent mida i propietats de superfície: NPs d'òxid de ferro (SPIONs) recobertes de citrat i d'albúmina sèrica bovina (BSA), i NPs d'or recobertes de citrat de dos mides diferents (10 nm i 150 nm). Hem estudiat les interaccions nano-bio en C. elegans amb especial atenció en les propietats dels materials in vivo incloent el seu estat, ingesta, localització, efectes biològics i mecanismes moleculars. Amb aquest propòsit, hem combinat assajos de toxicitat, tècniques de ciència de materials i d'imatge, i anàlisi de l'expressió gènica. També hem investigat la influència de la composició de les NPs, del seu recobriment superficial i de la seva mida in vivo, i comparat els nostres resultats amb estudis previs. És destacable el fet d'haver pogut realitzar totes aquestes avaluacions biològiques en el mateix laboratori on hem sintetitzat i caracteritzat les partícules gracies als avantatges experimentals de C. elegans. Així doncs, aquesta tesi valida l'ús de C. elegans com a un model animal senzill per avaluar NPs en les etapes inicials de desenvolupament. A través de tècniques de microscòpia, vam veure que les NPs entraven al cos del cuc durant el procés d'alimentació i s'acumulaven seguint patrons particulars segons les propietats de les NPs. Les NPs d'or no eren internalitzades per les cèl·lules intestinals de C. elegans, mentre que vam poder identificar NPs d'òxid de ferro en el compartiment lisosomal d'aquestes cèl·lules. En cap cas vam detectar translocació de NPs a òrgans secundaris, ni entrades per altres vies (ex. dermal) o orificis del cos. Per caracteritzar el perfil toxicològic de les NPs, vam estudiar diferents paràmetres toxicològics letals i sub-letals. Vam observar que la LD50 de les NPs d'òxid de ferro casi doblava (600 µg/ml) el valor de les NPs d'or de 11 nm (350 µg/ml), cosa que indica que les NPs d'òxid de ferro són més biocompatibles que les d'or. Les SPIONs recobertes de BSA van afectar l'estat de C. elegans menys que les recobertes de citrat, suggerint un efecte protector pel recobriment del BSA. De manera similar, vam veure que les NPs d'or de 150 nm eren menys tòxiques que les de 11 nm, cosa que indica que la mida de les NPs és rellevant pels seus efectes in vivo. L'ús de tècniques fisicoquímiques ens va permetre quantificar la ingesta de NPs per part del cuc i ens va permetre també caracteritzar l'estat de les NPs en C. elegans. Dins de l'animal, les SPIONs mantenien les seves propietats magnètiques inicials però eren parcialment degradades en l'intestí, especialment les recobertes de citrat, amb el conseqüent alliberament de ions ferro. Les NPs d'or van resultar més resistents a la degradació però vam observar cert grau d'agregació, que era reversible i depenia de la regió de l'intestí on es localitzaven les NPs. Mitjançant experiments in vitro, vam confirmar que el pH i la presència de biomolècules en l'intestí pot contribuir a determinar l'estat de les NPs in vivo. D'altra banda, vam estudiar i identificar mecanismes moleculars afectats per l'exposició a NPs a través de tècniques genètiques. Vam observar que les NPs alteraven els mecanismes d'estrès oxidatiu i de detoxificació de metalls, cosa que suggereix que estan implicats en la resposta de C. elegans a les NPs. En el cas de les SPIONs, vam detectar efectes depenent del recobriment: mentre que les partícules recobertes de citrat afectaven el processament d'informació ambiental i genètica, les recobertes de BSA afectaven el funcionament metabòlic. En conjunt, creiem que el tractament amb NPs és capaç d'activar mecanismes d'estrès general i també rutes específiques que depenen de les propietats de les NPs. En resum, aquesta tesi ha contribuït a: i) ampliar el ventall de tècniques utilitzades per caracteritzar interacciones nano-bio en C. elegans; ii) una avaluació sistemàtica i extensa de les interacciones entre NPs y C. elegans, des de la síntesi del material fins l'anàlisi dels mecanismes moleculars; i iii) estudiar la influencia de las propietats de les NPs en els seus efectes in vivo.En esta tesis hemos usado el modelo animal Caenorhabditis elegans (C. elegans) como sistema biológico vivo en el que evaluar nanopartículas (NPs) para aplicaciones biomédicas. En concreto, hemos evaluado el comportamiento de dos tipos de partículas inorgánicas con diferentes tamaños y propiedades de superficie: NPs de óxido de hierro (SPIONs) recubiertas de citrato y de albúmina sérica bovina (BSA), y NPs de oro recubiertas de citrato de dos tamaños distintos (10 nm y 150 nm). Hemos estudiado las interacciones nano-bio en C. elegans con especial atención en las propiedades de los materiales in vivo incluyendo su estado, ingestión, localización, efectos biológicos y mecanismos moleculares. Con esta finalidad, hemos combinado ensayos de toxicidad, técnicas de ciencia de materiales y de imagen, y análisis de la expresión génica. También hemos investigado la influencia de la composición de las NPs, de su recubrimiento superficial y de su tamaño in vivo, y comparado nuestros resultados con estudios previos. Es destacable el hecho de haber podido realizar todas estas evaluaciones biológicas en el mismo laboratorio donde hemos sintetizado y caracterizado las partículas gracias a las ventajas experimentales de C. elegans. De este modo, esta tesis valida el uso de C. elegans como un modelo animal simple para evaluar NPs en las etapas iniciales de desarrollo. A través de técnicas de microscopía, vimos que las NPs entraban al cuerpo del gusano durante el proceso de alimentación y se acumulaban siguiendo patrones particulares según las propiedades de las NPs. Las NPs de oro no eran internalizadas por las células intestinales de C. elegans, mientras que detectamos SPIONs en el compartimento lisosomal de estas células. En ningún caso detectamos translocación de NPs a órganos secundarios, ni entrada por otras vías (ej. dermal) u orificios del cuerpo. Para caracterizar el perfil toxicológico de las NPs, estudiamos diferentes parámetros toxicológicos letales y sub-letales. Observamos que la LD50 de las SPIONs casi doblaba (600 µg/ml) el valor de las NPs de oro de 11 nm (350 µg/ml), lo cual indica que las SPIONs son más biocompatibles que las de oro. Las BSA-SPIONs afectaron el estado de C. elegans menos que las C-SPIONs, sugiriendo un efecto protector del recubrimiento de BSA. De forma similar, vimos que las NPs de oro de 150 nm eran menos tóxicas que las de 11 nm, lo cual indica que el tamaño de las NPs es relevante para sus efectos in vivo. El uso de técnicas fisicoquímicas nos permitió cuantificar la ingesta de NPs por parte del gusano y nos permitió también caracterizar el estado de las NPs en C. elegans. Dentro del animal, las SPIONs mantenían sus propiedades magnéticas iniciales pero eran parcialmente degradadas en el intestino, especialmente las recubiertas de citrato, con la consiguiente liberación de iones hierro. Las NPs de oro resultaron más resistentes a la degradación pero observamos cierto grado de agregación, que era reversible y dependía de la región del intestino donde se localizaban las NPs. Mediante experimentos in vitro, confirmamos que el pH y la presencia de biomoléculas en el intestino puede contribuir a determinar el estado de las NPs in vivo. Por otro lado, estudiamos e identificamos mecanismos moleculares afectados por la exposición a NPs usando técnicas genéticas. Observamos que las NPs alteraban los mecanismos de estrés oxidativo y de detoxificación de metales, lo cual sugiere que están implicadas en la respuesta de C. elegans a las NPs. En el caso de las SPIONs, detectamos efectos dependientes del recubrimiento: mientras que las partículas recubiertas de citrato afectaban el procesamiento de información ambiental y genética, las recubiertas de BSA afectaban el funcionamiento metabólico. En conjunto, creemos que el tratamiento con NPs es capaz de activar mecanismos de estrés general y también rutas específicas que dependen de las propiedades de las NPs. En resumen, esta tesis ha contribuido a: i) extender el abanico de técnicas usadas para caracterizar interacciones nano-bio en C. elegans; ii) una evaluación sistemática y extensa de las interacciones entre NPs y C. elegans, desde la síntesis del material hasta el análisis de mecanismos moleculares; y iii) estudiar la influencia de las propiedades de las NPs en sus efectos in vivo.In this thesis, we have used the simple model organism Caenorhabditis elegans (C. elegans) as an in vivo biological system to screen nanoparticles (NPs) proposed for biomedical uses. In particular, we have assessed the behaviour of two types of inorganic particles with different size and surface properties: iron oxide NPs (SPIONs) coated with citrate and bovine serum albumin (BSA), and citrate coated gold nanoparticles of two different diameters (11 nm and 150 nm). We have studied their nano-bio interactions in C. elegans with special focus on the material's properties in vivo including their status, uptake, fate, biological effects and molecular mechanisms. To this end, we have combined toxicity tests, materials science and imaging techniques, and gene expression analysis. We have also investigated the influence of NP composition, coating and size in vivo, and compared our results with previous studies. Remarkably, we have been able to perform this biological evaluation in the synthetic laboratory where the particles were synthesized and characterised due to the advantageous experiments features of C. elegans. Therefore, our work sets up C. elegans as a simple animal model to evaluate NPs in the initial stages of development. By microscopy techniques, we found that NPs entered the body of the worm during feeding and accumulated in the intestine with particular patterns depending on the NP properties. Gold NPs were not internalized by the C. elegans intestinal cells, while iron oxide NPs were identified in the lysosomal compartment of these cells. We did not detect translocation of NPs to secondary organs, either entrance by other routes (i.e. dermal) or body openings. Lethal and sub-lethal endpoints were analysed to characterise the toxicological profile of the NPs. We found that the LD50 of iron oxide NPs almost doubled (600 µg/ml) the value for 11-nm gold NPs (350 µg/ml) indicating the superior biocompatibility of the former. BSA-coated SPIONs affected to a lesser extent the behaviour of C. elegans than citrate-coated SPIONs, suggesting a protective effect of the BSA coating. Similarly, 150-nm gold NPs appeared less toxic than their smaller counterparts, indicating that NP size is relevant to their in vivo effects. The use of physicochemical techniques allowed us to quantify the ingestion of NPs by C. elegans and, interestingly, we could also characterise NP status inside C. elegans. We found that iron oxide NPs maintained their initial magnetic properties but were partially degraded inside the intestine, especially the citrate-coated particles, with the consequent release of iron ions. Gold NPs appeared resistant to degradation but showed some degree of aggregation, which was reversible and depended on the specific area of the gut. We confirmed in vitro that the pH and presence of biomolecules in the intestine could contribute to determine the status of the NPs in vivo. We also investigated potential molecular mechanisms affected by NP exposure using genetic tools. We observed that NP treatment disrupted the oxidative stress and metal detoxification pathways, suggesting that they might be involved in the response of C. elegans to NPs. In the case of iron oxide NPs, we found coating-dependent effects: citrate-coated particles affected the processing of environmental and genetic information, while BSA-coated SPIONs affected metabolic mechanisms. All in all, we believe that NP treatment can trigger general stress mechanisms and also specific pathways depending on the NP properties. In conclusion, this thesis contributes to: i) extend the toolkit of techniques used for the characterisation of nano-bio interactions in C. elegans; ii) a systematic and comprehensive evaluation of the interaction of NPs in C. elegans, from the materials' synthesis to the analysis of molecular pathways; and iii) study the influence of NP properties on their in vivo effects

Pth reloaded: a new evolutionary perspective

The parathyroid hormone (PTH) family is a group of structurally-related secreted peptides involved in bone mineral homeostasis and multitude of developmental processes in vertebrates. These peptides mediate actions through PTH receptors (PTHRs), which belong to the transmembrane G protein-coupled receptor group. To date, genes encoding for PTH and PTHR have only been identified in chordates, suggesting that this signaling pathway may be an evolutionary innovation of our phylum. In vertebrates, we found up to six PTH and three PTHR different paralogs, varying in number between mammals and teleost fishes due to the different rounds of whole-genome duplication and specific gene losses suffered between the two groups of animals. The diversification of the PTH gene family has been accompanied by both functional divergence and convergence, making sometimes difficult the comparison between PTH peptides of teleosts and mammals. Here, we review the roles of all Pth peptides in fishes, and based on the evolutionary history of PTH paralogs, we propose a new and simple nomenclature from PTH1 to PTH4. Moreover, the recent characterization of the Pth4 in zebrafish allows us to consider the prominent role of the brain-to-bone signaling pathway in the regulation of bone development and homeostasis. Finally, comparison between PTH peptides of fish and mammals allows us to discuss an evolutionary model for PTH functions related to bone mineral balance during the vertebrate transition from an aquatic to a terrestrial environment

Latent Classes for the Treatment Outcomes in Women with Gambling Disorder and Buying/Shopping Disorder

Background: The risk for behavioral addictions is rising among women within the general population and in clinical settings. However, few studies have assessed treatment effectiveness in females. The aim of this work was to explore latent empirical classes of women with gambling disorder (GD) and buying/shopping disorder (BSD) based on the treatment outcome, as well as to identify predictors of the different empirical groups considering the sociodemographic and clinical profiles at baseline. Method: A clinical sample of n = 318 women seeking treatment for GD (n = 221) or BSD (n = 97) participated. Age was between 21 to 77 years. Results: The four latent-classes solution was the optimal classification in the study. Latent class 1 (LT1, good progression to recovery) grouped patients with the best CBT outcomes (lowest risk of dropout and relapses), and it was characterized by the healthiest psychological state at baseline, the lowest scores in harm avoidance and self-transcendence, and the highest scores in reward dependence, persistence, self-directedness and cooperativeness. Latent classes 3 (LT3, bad progression to drop-out) and 4 (LT4, bad progression to relapse) grouped women with the youngest mean age, earliest onset of the addictive behaviors, and worst psychological functioning. Conclusions: GD and BSD are complex conditions with multiple interactive causes and impacts, which need wide and flexible treatment plans. Specific interventions should be designed according to the specific profiles of women for achieving early inclusion, retention and well-maintained long-term effects

e-Estesia: A Serious Game for Reducing Arousal, Improving Emotional Regulation and Increasing Wellbeing in Individuals with Gambling Disorder

Gambling disorder (GD) is associated with deficits in emotion regulation and impulsivity-related personality traits. In recent years there has been an increase in the use of serious games (SG) to address these factors with positive results. The aim of this study was to analyze the efficacy of the intervention with a new SG (e-Estesia), as an adjunct to a CBT intervention for GD. The sample comprised two groups (experimental group (n = 40) and control group (n = 64)) of patients with GD diagnosis. Both groups received 16 weekly CBT sessions and, concurrently, only the experimental group received 15 additional sessions with e-Estesia. Pre-post treatment with e-Estesia administered in both groups were: DSM-5 Criteria, South Oaks Gambling Screen, Symptom Checklist-Revised and measure of relapses, dropout and compliance of treatment. As regards the experimental group were also administered: Difficulties in Emotion Regulation Scale, Emotion Regulation Questionnaire, and Impulsive Behavior Scale. No statistically significant differences in the general psychopathological state, emotion regulation or impulsivity were found when comparing the groups. However, patients enrolled in the e-Estesia intervention had significantly less relapses and better indicators of treatment compliance than the control group. Considering these results, the use of complementary tools such as SG are useful for addressing GD

Executive functioning among female pathological gambling and bulimia nervosa patients: preliminary findings

Shared vulnerabilities have been described across disorders of impulse control, including pathological gambling (PG) and bulimia nervosa (BN). Our aim was to compare the executive functioning of PG and BN females in order to confirm their similarity at a neurocognitive level. A total of 15 BN females, 15 PG females, and 15 healthy control (HC) females were administered the Wisconsin Card Sorting Test (WCST) and the Stroop Color and Word Test. Analysis of covariance adjusted for age and education was conducted to compare groups. PG showed the greatest impairment, that is, the highest percentage of WCST perseverative errors (p = .023), the lowest percentage of conceptual-level responses (p = .034), and the highest number of total trials administered (p = .021), while BN showed the highest percentage of WCST nonperseverative errors (p = .003). Both BN and PG females demonstrated executive dysfunction relative to HCs but different specific correlates (i.e., greater vulnerability to distraction in BN, but more cognitive inflexibility in PG)

Gambling disorder duration and cognitive behavioural therapy outcome considering gambling preference and sex

Gambling Disorder (GD) is a behavioural addiction that leads to high level of clinical distress and, in general, it is characterized by enduring symptomatology that presents high rates of chronicity. However, there is high variability of illness duration among patients who seek treatment for GD. Previous studies reported mixed results about the relevance of illness duration in GD treatment outcome. However, there are different profiles of patients who are diagnosed with GD. For this reason, this study aimed to evaluate the effect of illness duration in the treatment outcome of different profiles of GD patients according to their gambling preference and sex. The sample were 1699 patients diagnosed with GD. All patients received cognitive-behavioural therapy in a group format. Treatment outcome was evaluated in terms of relapsing to gambling behaviours and dropout from treatment. Results showed higher probability of poor outcome in the first years of the disorder for strategic gambling compared to non-strategic or mixed forms of gambling. Moreover, women also showed higher probability of poor outcomes than men since the first stages of the disorder. This study draws attention to the relevance of illness duration in the treatment outcome of specific profiles of GD patients. In particular, patients who presented a preference for strategic forms of gambling and women who are diagnosed with GD would have a higher risk of poor treatment outcomes since the first stages of the disorder. These results highlight the importance of an early intervention in these patients in order to prevent the chronicity of the disorder

Common and differential risk factors behind suicidal behavior in patients with impulsivity-related disorders: the case of bulimic spectrum eating disorders and gambling disorder

Background and aims: Mental disorders with high levels of impulsivity such as bulimic spectrum eating disorders (BSED) and gambling disorder (GD) are associated with high risk of suicidal behavior. The aim of the present study was to identify the common and differential vulnerability factors behind suicide attempts in a sample of patients with BSED compared to patients with GD. Methods: A total of 6,077 adults who sought treatment and met criteria either for BSED (n 5 2,391) or GD (n 5 3,686) were assessed at a specialized hospital unit. Personality traits, psychopathological symptomatology, lifetime history of suicide attempts and socio-demographic variables were evaluated. Results: The prevalence of suicide attempts was higher for BSED patients (26.2%) compared to GD patients (7.1%) being anorexia nervosa (Binge/Purge type) and bulimia nervosa the most affected subtypes. In the predictive model, the transdiagnostic vulnerability factors with the highest contribution to the risk of suicidal behavior both in BSED and GD were unemployment, early age of onset of the disorder, worse psychopathological state, and self-transcendence personality trait. However, specific risk factors for suicidal acts were identified in each disorder: longer duration of the disorder, lower education levels and reward dependence were exclusively associated with BSED while female gender, older age, and higher harm avoidance were associated with GD. Discussion: Patients with GD and BSED share certain vulnerability factors although certain factors are exclusive to each disorder. Conclusions: Interventions need to pay special attention to both common and specific vulnerability factors to mitigate the risk of suicidal acts in these disorders