Encapsulation of biologically active agents in emulsions stabilized by natural polymers

El uso de nanotransportadores para la encapsulación de diferentes moléculas ha sido explorado a lo largo de los últimos años para múltiples aplicaciones. Ha sido utilizado por ejemplo para la vehiculización de moléculas hidrofóbicas en medios acuosos (y de hidrofílicas en medios oléicos), la protección de moléculas sensibles o la inmovilización de diferentes moléculas en soportes.Entre los diferentes tipos de nanotransportadores que han sido desarrollados, las nanocápsulas poliméricas han atraído la atención de multitud de investigadores debido a sus grandes ventajas. En esta tesis, nanoemulsiones recubiertas de polisacáridos han sido desarrolladas para su utilización como nanotransportadores de diferentes moléculas. Gracias a su versatilidad, estas nanocápsulas permiten la encapsulación de moléculas hidrofóbicas en el interior aceitoso, mientras que las moléculas hidrofílicas pueden ser atrapadas en la recubierta polimérica. Además, los componentes de las nanocápsulas pueden ser modificados a demanda de la aplicación final.Las nanocápsulas desarrolladas han demostrado ser muy efectivas para la encapsulación de antibióticos como bedaquilina y daptomicina para su uso en el tratamiento de infecciones causadas por Mycobacterium tuberculosis y Staphylococcus aureus resistente a la meticilina (SARM), respectivamente. Han sido utilizadas también en un estudio exploratorio para la encapsulación del fármaco disulfiram y su utilización alternativa en el tratamiento del cáncer de páncreas e infecciones causadas por SARM. También han mostrado gran capacidad como soportes para la inmovilización de la enzima cloroperoxidasa así como la encapsulación de partículas magnéticas. Por último, también han sido desarrolladas nanocápsulas para la encapsulación de nanopartículas de vanadato de holmio para su utilización como agentes de contraste.Todas las nanocápsulas desarrolladas han sido recubiertas con el polímero adecuado teniendo en cuenta la aplicación final y las necesidades que se debían cubrir. Tanto el quitosano como el alginato y el xantano han sido utilizados para este propósito de manera satisfactoria. Se han realizado las caracterizaciones fisicoquímicas así como las pruebas de eficacia necesarias para cada aplicación.En todos los casos, estas nanocápsulas han demostrado tener una gran versatilidad como agentes nanotransportadores de diferentes fármacos, enzimas y nanopartículas así como ser capaces de modificar su superficie con diferentes polímeros para conseguir los requerimientos de la aplicación final.The use of nanocarriers for the encapsulation of different molecules has been explored over the recent years for multiple applications. They have been used for the vehiculization of hydrophobic molecules in aqueous media (or hydrophilic ones in oily media), protection of sensitive molecules or immobilization of different molecules in supports. Among the different nanocarriers that have been developed, polymeric nanocapsules have attracted the interest of many researchers thanks to their great advantages. In this thesis, polysaccharide coated nanoemulsions have been developed as potential nanocarriers for the encapsulation of different molecules. Thanks to their versatility, these nanocarriers allow the encapsulation of hydrophobic in the oily core, while hydrophilic ones can be entrapped in the polymer coating. Besides, the components of the nanocapsules can be modified upon the needs of the final application. All the developed nanocapsules were coated with the adequate polymer depending on the final application and the characteristics that needed to be achieved. Both chitosan, alginate and xanthan were used successfully. The developed nanocapsules have proven to be very effective for the encapsulation of antibiotics such as bedaquiline and daptomycin used the treatment of Mycobacterium tuberculosis and Methicillin-resistant Staphylococcus aureus (MRSA) infections, respectively. An exploratory research has been also carried out to encapsulate the drug disulfiram for its repurposing for the treatment of pancreatic cancer and MRSA infections. The developed nanocapsules have shown great capability as immobilizing agents of the enzyme chloroperoxidase and the encapsulation of magnetic nanoparticles (NP). They have also been able to encapsulate holmium vanadate NP for their use as contrast agents. Physicochemical characterization as well as efficacy test for the different purposes were performed in all the cases. For all the tested applications, the developed nanocapsules showed great versatility for their use as nanocarriers of many different drugs, enzymes and nanoparticles as well as being able to modify the surface with different polymers to meet the application requirements.<br /

Encapsulation of biologically active agents in emulsions stabilized by natural polymers

Programa de Doctorado en Bioquímica y Biología Molecular.[ES] El uso de nanotransportadores para la encapsulación de diferentes moléculas ha sido explorado a lo largo de los últimos años para múltiples aplicaciones. Ha sido utilizado por ejemplo para la vehiculización de moléculas hidrofóbicas en medios acuosos (y de hidrofílicas en medios oléicos), la protección de moléculas sensibles o la inmovilización de diferentes moléculas en soportes. Entre los diferentes tipos de nanotransportadores que han sido desarrollados, las nanocápsulas poliméricas han atraído la atención de multitud de investigadores debido a sus grandes ventajas. En esta tesis, nanoemulsiones recubiertas de polisacáridos han sido desarrolladas para su utilización como nanotransportadores de diferentes moléculas. Gracias a su versatilidad, estas nanocápsulas permiten la encapsulación de moléculas hidrofóbicas en el interior aceitoso, mientras que las moléculas hidrofílicas pueden ser atrapadas en la recubierta polimérica. Además, los componentes de las nanocápsulas pueden ser modificados a demanda de la aplicación final. Las nanocápsulas desarrolladas han demostrado ser muy efectivas para la encapsulación de antibióticos como bedaquilina y daptomicina para su uso en el tratamiento de infecciones causadas por Mycobacterium tuberculosis y Staphylococcus aureus resistente a la meticilina (SARM), respectivamente. Han sido utilizadas también en un estudio exploratorio para la encapsulación del fármaco disulfiram y su utilización alternativa en el tratamiento del cáncer de páncreas e infecciones causadas por SARM. También han mostrado gran capacidad como soportes para la inmovilización de la enzima cloroperoxidasa así como la encapsulación de partículas magnéticas. Por último, también han sido desarrolladas nanocápsulas para la encapsulación de nanopartículas de vanadato de holmio para su utilización como agentes de contraste. Todas las nanocápsulas desarrolladas han sido recubiertas con el polímero adecuado teniendo en cuenta la aplicación final y las necesidades que se debían cubrir. Tanto el quitosano como el alginato y el xantano han sido utilizados para este propósito de manera satisfactoria. Se han realizado las caracterizaciones fisicoquímicas así como las pruebas de eficacia necesarias para cada aplicación. En todos los casos, estas nanocápsulas han demostrado tener una gran versatilidad como agentes nanotransportadores de diferentes fármacos, enzimas y nanopartículas así como ser capaces de modificar su superficie con diferentes polímeros para conseguir los requerimientos de la aplicación final.[EN] The use of nanocarriers for the encapsulation of different molecules has been explored over the recent years for multiple applications. They have been used for the vehiculization of hydrophobic molecules in aqueous media (or hydrophilic ones in oily media), protection of sensitive molecules or immobilization of different molecules in supports. Among the different nanocarriers that have been developed, polymeric nanocapsules have attracted the interest of many researchers thanks to their great advantages. In this thesis, polysaccharide coated nanoemulsions have been developed as potential nanocarriers for the encapsulation of different molecules. Thanks to their versatility, these nanocarriers allow the encapsulation of hydrophobic in the oily core, while hydrophilic ones can be entrapped in the polymer coating. Besides, the components of the nanocapsules can be modified upon the needs of the final application. All the developed nanocapsules were coated with the adequate polymer depending on the final application and the characteristics that needed to be achieved. Both chitosan, alginate and xanthan were used successfully. The developed nanocapsules have proven to be very effective for the encapsulation of antibiotics such as bedaquiline and daptomycin used the treatment of Mycobacterium tuberculosis and Methicillin-resistant Staphylococcus aureus (MRSA) infections, respectively. An exploratory research has been also carried out to encapsulate the drug disulfiram for its repurposing for the treatment of pancreatic cancer and MRSA infections. The developed nanocapsules have shown great capability as immobilizing agents of the enzyme chloroperoxidase and the encapsulation of magnetic nanoparticles (NP). They have also been able to encapsulate holmium vanadate NP for their use as contrast agents. Physicochemical characterization as well as efficacy test for the different purposes were performed in all the cases. For all the tested applications, the developed nanocapsules showed great versatility for their use as nanocarriers of many different drugs, enzymes and nanoparticles as well as being able to modify the surface with different polymers to meet the application requirements.Peer reviewe

Controlling properties and cytotoxicity of chitosan nanocapsules by chemical grafting

The tunability of the properties of chitosan-based carriers opens new ways for the application of drugs with low water-stability or high adverse effects. In this work, the combination of a nanoemulsion with a chitosan hydrogel coating and the following poly (ethylene glycol) (PEG) grafting is proven to be a promising strategy to obtain a flexible and versatile nanocarrier with an improved stability. Thanks to chitosan amino groups, a new easy and reproducible method to obtain nanocapsule grafting with PEG has been developed in this work, allowing a very good control and tunability of the properties of nanocapsule surface. Two different PEG densities of coverage are studied and the nanocapsule systems obtained are characterized at all steps of the optimization in terms of diameter, Z potential and surface charge (amino group analysis). Results obtained are compatible with a conformation of PEG molecules laying adsorbed on nanoparticle surface after covalent linking through their amino terminal moiety. An improvement in nanocapsule stability in physiological medium is observed with the highest PEG coverage density obtained. Cytotoxicity tests also demonstrate that grafting with PEG is an effective strategy to modulate the cytotoxicity of developed nanocapsules. Such results indicate the suitability of chitosan as protective coating for future studies oriented toward drug delivery.Authors would like to acknowledge the public funding from Fondo Social de la DGA (grupos DGA), Ministerio de la Economía y Competitividad del Gobierno de España for the public founding of ProyectosI+D+i—Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad (project n. SAF2014-54763-C2-2-R), the European Seventh Framework Program (NAREB Project 604237), LLP/Erasmus fellowship 2013/2014, INA fellowship “Iniciación a la Investigación” 2014 and 2015, and the European Union’s Horizon 2020 research and innovation program for MCSA Fellowship (Grant Agreement No. 660228).We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Nanoemulsion-based chitosan nanocapsules as antibiotic delivery system

Resumen del trabajo presentado al IMI Translocation - Novel approaches to fight bacteria: "From understanding the crossing of membrane barrier to the development of new nanotechnology-based drugs", celebrado en Bremen (Alemania) del 10 al 14 de julio de 2016.-- et al.The combination of nanoemulsion method and chitosan coating presented in this work has been proved to be a promising strategy to obtain a versatile multi-pocket nanocarrier. The use of antibiotics produces important side effects, thus reducing the dose of administration and targeting the infection sites are issues of important concern. Nanotechnology represents an important tool to improve the residence and blood circulation time of the delivered drugs so allowing the reduction of the dose and possibly the side effects. In the past few decades, many kinds of nanocarriers have been developed for delivery and targeting of therapeutic or diagnostic agents for medical treatments, thanks to some important advantages that they offer depending on their physicochemical properties. Different antibiotics active both against MRSA (Methicillin-resistant Staphylococcus aureus) and MRTB (Multi-resistant Mycobacterium tuberculosis) have been successfully encapsulated in nanoemulsion-based chitosan nanocapsules. In particular, bedaquiline, amikacin, active against Mycobacterium tuberculosis, and daptomycin, against Staphylococcus aureus. The encapsulation of the selected antibiotics is reported, in terms of optimization of the drug loading and encapsulation efficiency of the process. The systems obtained have been fully characterized and their antimicrobial effect has been tested, proving the maintenance of the drug activity once the antibiotic is encapsulated.Peer Reviewe

Effective in vitro photokilling by cell-adhesive gold nanorods

Upon excitation of their localized surface plasmon resonance (LSPR) band, gold nanorods (AuNRs) show a characteristic light-to-heat transduction, a useful and versatile property for a range of biomedical applications such as photothermal therapy, drug delivery, optoacoustic imaging and biosensing, among others. Nanoparticle (NP)-mediated photothermal therapy (PTT) rests on the ability of nanomaterials to convert light energy into heat and can currently be considered as a promising method for selectively destroying tumor cells by (photo)-thermoablation. One inherent limitation to NP-mediated PTT is that the nanoparticles must arrive at the site of action to exert their function and this typically involves cellular internalization. Here we report the use of the Keggin-type polyoxometalate (POM) phosphotungstic acid (PTA) as an inorganic gelling agent for the encapsulation of plasmonic gold nanorods (AuNRs) inside a biocompatible and cell-adhesive chitosan hydrogel matrix. These functional sub-micrometric containers are non-cytotoxic and present the ability to adhere to the cytoplasmic membranes of cells avoiding any need for cellular internalization, rendering them as highly efficient thermoablating agents of eukaryotic cells in vitro.Financial support by the Fundación General CSIC (SM, Programa ComFuturo), Fondo Social Europeo-Gobierno de Aragón and Ministerio de Educación, Cultura y Deportes (ÁA for FPU grant FPU014/06249 and SG-E for FPU grant FPU15/04482) is gratefully acknowledged.Peer reviewe

Magnetic separation and high reusability of chloroperoxidase entrapped in multi polysaccharide micro-supports

Trabajo presentado a la IV Reunión de Jóvenes Investigadores en Coloides e Interfases, celebrada en Córdoba del 7 al 9 de febrero de 2018.Enzyme immobilization on magnetic supports represents a great advantage for the industrial application of enzymatic catalysis since it allows an easy recovery of the catalyst, avoiding any contamination of the product by residual enzyme. Iron oxide nanoparticles are very useful for this purpose. They are frequently coated with polymers to diminish the interaction between the magnetic cores themselves, improving colloidal stability of the support, and preventing any interaction with the environment that would affect both support properties and enzyme stability. In this work different magnetic micro-supports, based on polydopammine-coated iron oxide nanoparticles with a multi polysaccharidic shell, have been developed. These supports have been used to immobilize chloroperoxidase, a very interesting enzyme, able to catalyze many reactions of large-scale interest, but whose application is limited by its sensitivity to reaction conditions. The multi polysaccharidic shells of the supports were obtained through a combination of chitosan and alginate, that were chosen for their pH-dependent properties and for their already reported stabilizing effect on this enzyme, especially in the case of chitosan. An in-depth analysis of physicochemical properties of all the developed magnetic supports, especially in terms of their stability under reaction conditions, allowed to find the one that showed the best reusability of the catalyst.Peer Reviewe

Magnetic separation and high reusability of chloroperoxidase entrapped in multi polysaccharide micro-supports

Resumen del trabajo presentado al 37th Spring Meeting of the European Materials Research Society (E-MRS), celebrado del 27 al 31 de mayo de 2019 en Niza (Francia).The World Commission on Environment and Development introduced the term sustainable development, indicating the present need of modern industrial processes to optimize the use of raw materials, reduce waste and avoid the use of toxic molecules. Amongst the approaches used over the years to achieve this sustainability, biocatalysts, especially enzymes, have been in the spotlight due to their great properties. Sustainability of enzymatic catalysis is maintained through the whole cycle: from their production (living organisms) to the waste treatment. However, their present application at industrial scale is hampered by the high costs in their production that decrease cost-effectiveness of their application. Reutilization of the enzyme is therefore the tool to obtain more cost-effective and sustainable industrial processes. Immobilization of these biocatalysts allows an easy recovery of the material and protection from the reaction conditions in the different production steps. Nowadays, nanotechnology offers one of the most forefront approaches for enzyme immobilization. Magnetic nanoparticles allow an easy recovery of an immobilized enzyme using a simple magnet to separate the catalyst from the reaction product. To improve colloidal stability of the support, reduce interactions between the magnetic cores and prevent interactions with the environment that can affect both support and enzyme stability, a polymer coating is an easy and cheap approach. Using this approach, in this work we developed a hybrid, modular micro-support based on organic and inorganic nanocomponents. The easiness of tuning the composition of the support makes this system a potentially universal support for the immobilization of very different catalytic systems. Here we present the application of the developed micro-support for the immobilization of chloroperoxidase (CPO), an enzyme able to catalyze many reactions of large-scale interest. A multipolysaccharidic shell containing the immobilized enzyme and obtained through a combination of chitosan and alginate, biodegradable polymers from natural sources, was used to stabilize a nanoemulsion core in which magnetic nanoparticles were embedded. Microsupports obtained through different combinations of nanocomponents were characterized and tested in terms of their chemical stability under reaction conditions. An excellent reusability of the entrapped enzyme was observed opening the way to the immobilization of different catalytic systems and to the scale-up study in view of future industrial application

Magnetic separation and high reusability of chloroperoxidase entrapped in multi polysaccharide micro-supports

Enzyme immobilization on magnetic supports represents a great advantage for the industrial application of enzymatic catalysis since it allows an easy recovery of the catalyst, avoiding any contamination of the product by residual enzyme. Iron oxide nanoparticles are very useful for this purpose. Using a polymer to diminish the interaction between the magnetic cores themselves, can improve the colloidal stability of the support and prevent any interaction with the environment that would affect both support properties and enzyme stability. For this reason, in this work different magnetic micro-supports, based on polydopamine-coated iron oxide nanoparticles with a multi polysaccharide shell, have been developed. These supports have been used to immobilize chloroperoxidase, a very interesting enzyme, able to catalyze many reactions of large-scale interest, but whose application is limited by its sensitivity to reaction conditions. The multi polysaccharide shells of the supports were obtained through a combination of chitosan and alginate. An in-depth analysis of physicochemical and catalytic properties of all the developed magnetic supports is reported. CPO was successfully immobilized with an efficiency of entrapment between 92% and 100% in the case of supports with chitosan in the interior or outer shell respectively. A very good chemical stability of the support under reaction conditions was observed in the case of an interior shell of alginate and an outer coating of chitosan, together with an excellent reusability of the immobilized enzyme, that was recycled to catalyze up to 25 consecutive reaction cycles.Authors would like to acknowledge the public funding from Fondo Social de la DGA (grupos DGA). S. García-Embid and F. Di Renzo respectively acknowledge also Ministerio de Educación Cultura y Deporte (fellowship FPU15/04482) and the PROGRAMME LLP/ERASMUS 2015/16 (student placement).Peer reviewe

Innovative Inkjet printed encapsulation of gold nanoparticles inside celladhesive chitosan hydrogels for oral administration

Resumen del trabajo presentado al 37th Spring Meeting of the European Materials Research Society (E-MRS), celebrado del 27 al 31 de mayo en Niza (Francia)

Nanotransportadores basados en nanocápsulas poliméricas

Resumen del póster presentado a la 8ª Jornada de Jóvenes Investigadores (Química y Física) de Aragón, celebrada en Zaragoza el dia 22 de noviembre de 2018.En las últimas décadas, se han desarrollado múltiples tipos de nanopartículas para el transporte de agentes terapéuticos y de diagnóstico debido a las importantes ventajas que ofrecen dependiendo de sus propiedades físicoquímicas. La línea de investigación del grupo está enfocada en el desarrollo de nanotransportadores biocompatibles para aplicaciones biomédicas y biotecnológicas. En particular, los nanotransportadores sintetizados para este trabajo son nanocápsulas formadas por un núcleo lipídico anfifílico, que permite la encapsulación tanto de moléculas hidrofóbicas como hidrofílicas, rodeado por un recubrimiento polimérico que aumenta la estabilidad y protege la molécula encapsulada de la degradación. Para el recubrimiento polimérico se ha utilizado quitosano, que es un polisacárido biocompatible rico en grupos amino, lo que hace que sea muy fácil funcionalizar las nanocápsulas con otras moléculas para modificar sus propiedades según las necesidades. El material obtenido ha sido caracterizado en términos de tamaño (100-200 nm de diámetro medio) y potencial Z (positivo). Hasta la fecha se ha logrado encapsular moléculas activas, como antibióticos, ácidos nucleicos y proteínas, moléculas fluorescentes y nanopartículas inorgánicas.Peer Reviewe