Funcionalización de nanopartículas con marcadores biológicos e impregnación de matrices porosas a partir de extractos de hojas de mango

En los últimos años se ha producido un gran aumento en el interés de dos campos como son los nutracéuticos, sustancias de origen natural con propiedades biológicas activas beneficiosas para la salud y con capacidad preventiva y/o terapéutica definida, y la tecnología de nanopartículas, partículas con un tamaño entre 1-100 nm que presentan una serie de propiedades particulares debido a su tamaño, como una mayor biodisponibilidad. Por otro lado, si también se desea disponer de un sistema portador de estas partículas, para conseguir sistemas de liberación controlada, estas nanopartículas se pueden depositar en matrices sólidas porosas. La presente Tesis Doctoral está centrada en integrar estos dos novedosos campos de investigación mediante la obtención de extractos y nanopartículas de nutracéuticos con capacidad altamente antioxidante a partir de hojas de mango y su posible posterior aplicación en biomedicina, ya sea junto a marcadores biológicos o siendo impregnadas en distintas matrices porosas de utilidad en aplicaciones médicas. Con el fin de conseguir estos objetivos se ha utilizado la tecnología supercrítica. La Extracción Supercrítica (ESC), para obtener los extractos; la técnica Supercritical Antisolvent Extraction (SAS o SAE) para obtener nanopartículas a partir de dichos extractos naturales y la técnica Supercritical Solvent Impregnation (SSI) para la impregnación/deposición de extractos o partículas en matrices sólidas porosas. Además, las condiciones usadas de presión (100-300 bar) o temperatura (35-55 ºC) en estas técnicas con CO2 supercrítico no comprometen la estabilidad física o las capacidades de los principios activos utilizados, en su mayoría polifenoles termolábiles, al ser relativamente bajas. En el caso de la precipitación o funcionalización de los extractos de hoja de mango se ha seleccionado la fluoresceína como sustancia trazadora, la cual es posible seguir mediante fluorescencia en sus posibles aplicaciones médicas. Se ha estudiado tanto la precipitación de este trazador en solitario como la co-precipitación junto a las partículas de los extractos naturales, estudiando la influencia de distintos parámetros como presión, temperatura o flujo de CO2, entre otras. Además, se han realizado ensayos de viabilidad celular y eficacia de las partículas obtenidas mediante tecnología supercrítica frente al estrés oxidativo en macrófagos. Adicionalmente, se ha estudiado la precipitación y encapsulación de las nanopartículas junto a un anticuerpo, CXCL10, con el fin de ser utilizado como guía hacia el páncreas en futuras aplicaciones. Con respecto a la impregnación de los extractos o partículas en distintas matrices porosas mediante la técnica Supercritical Solvent Impregnation (SSI), se ha investigado la eficacia de la técnica y la influencia de distintas variables de operación en materiales como: apósitos para heridas, scaffolds poliméricos usados normalmente en ingeniería de tejidos y distintos tipos de sílice. Por último, se ha estudiado la viabilidad de los productos obtenidos como sistemas de liberación controlada en el tiempo, ya sea utilizando los compuestos presentes en los extractos etanólicos de hojas de mango o sustancias modelo como rutina o ácido gálico.In recent years, there has been a great increase in interest in two fields: nutraceuticals, substances of natural origin with active biological properties beneficial to health and defined preventive and/or therapeutic capacity, and nanoparticle technology, particles with a size between 1-100 nm that present a series of particular properties due to their size, such as greater bioavailability. On the other hand, if it is also desired to have a carrier system for these particles, in order to achieve controlled release systems, these nanoparticles can be deposited in porous solid matrices. The present Doctoral Thesis is focused on integrating these two novel fields of research by obtaining extracts and nanoparticles of nutraceuticals with high antioxidant capacity from mango leaves and their possible subsequent application in biomedicine, either together with biological markers or being impregnated in different porous matrices useful in medical applications. In order to achieve these objectives, supercritical technology has been used. Supercritical Extraction (ESC), to obtain the extracts; the Supercritical Antisolvent Extraction (SAS or SAE) technique to obtain nanoparticles from these natural extracts and the Supercritical Solvent Impregnation (SSI) technique for the impregnation/deposition of extracts or particles in porous solid matrices. Moreover, the pressure (100-300 bar) or temperature (35- 55 ºC) conditions used in these techniques with supercritical CO2 do not compromise the physical stability or the capacities of the used active ingredients, mostly thermolabile polyphenols, as they are relatively low. In the case of the precipitation or functionalization of mango leaf extracts, fluorescein has been selected as a tracer substance, which can be traced by fluorescence in its possible medical applications. Both the precipitation of this tracer alone and co-precipitation together with particles of natural extracts have been studied, investigating the influence of different parameters such as pressure, temperature or CO2 flow, among others. In addition, cell viability and efficacy tests of the particles obtained by supercritical technology against oxidative stress in macrophages have been carried out. In addition, the precipitation and encapsulation of the nanoparticles together with an antibody, CXCL10, has been studied in order to be used as a guide to the pancreas in future treatments. Regarding the impregnation of extracts or particles in different porous matrices using the Supercritical Solvent Impregnation (SSI) technique, the effectiveness of the technique and the influence of different operating variables on materials such as: wound dressings, polymeric scaffolds commonly used in tissue engineering and different types of silica have been investigated. Finally, the feasibility of the products obtained as time-controlled release systems has been studied, either using the compounds present in the ethanolic extracts of mango leaves or model substances such as rutin or gallic acid

Determining the Optimal Conditions for the Production by Supercritical CO2 of Biodegradable PLGA Foams for the Controlled Release of Rutin as a Medical Treatment

Poly(D,L,-lactide-co-glycolide) (PLGA) foam samples impregnated with rutin were successfully produced by supercritical foaming processes. A number of parameters such as pressure (80–200 bar), temperature (35–55 C), depressurization rate (5–100 bar/min), ratio lactide:glycolide of the poly(D,L,-lactide-co-glycolide) (50:50 and 75:25) were studied to determine their effect on the expansion factor and on the glass transition temperature of the polymer foams and their consequences on the release profile of the rutin entrapped in them. The impregnated foams were characterized by scanning electron microscopy, differential scanning calorimetry, and mercury intrusion porosimetry. A greater impregnation of rutin into the polymer foam pores was observed as pressure was increased. The release of rutin in a phosphate buffer solution was investigated. The controlled release tests confirmed that the modification of certain variables would result in considerable differences in the drug release profiles. Thus, five-day drug release periods were achieved under high pressure and temperature while the depressurization rate remained low.This research was funded by Spanish Ministry of Economy, Industry and Competitiveness: CTQ2017-86661-R

From olive leaves to spherical nanoparticles by one-step RESS process precipitation

In this work, spherical nanoparticles to be used in cosmetic, agro food or pharmaceutical industries have been directly precipitated from olives leaves in one-step RESS process. The leaves were brought into contact with supercritical CO2, and a fraction of the compounds from the flavone and flavonol families that can be found in the leaves were dissolved; then, by depressurizing the vessel, these compounds formed particles in the nanometer range. A complete factorial design was generated to thoroughly determine the influence from the main parameters on the RESS process with respect to the precipitated nanoparticles and their heterogeneity. Their antioxidant activity was also evaluated. Different pressures (250-350 bar), temperatures (60 and 100 degrees C), leaves sample weights (2 and 4 g) and cosolvent volumes, namely ethanol (9 and 18 mL), were studied as the main parameters that could affect the solvation and precipitation of the particle with active compounds in the leaves. Other parameters such as contact time (1 h) or nozzle size diameter (100 mu m) remained unchanged. The antioxidant activity was evaluated by means of the radical scavenging method using the radical 2,2-diphenyl-1-picrylhydrazole (DPPH). Spherical particles with diameters in the range of 55 nm to 4 mu m were obtained. Lower pressures and higher temperatures seemed to result in a reduction of the mean particle size. Greater volume of cosolvent is also recommended to reduce mean particle size. However, lower pressure, temperature and volume of cosolvent seems to promote a greater homogeneity of the particles. By means of chromatographic analyses, the main compounds, responsible for the antioxidant activity, such as oleuropein, quercetin or apigenin among others were identified

Espectroradiometría de campo del visible al infrarrojo térmico de muestras con características espectrales singulares

La calibración de sensores a bordo de satélite requiere medidas espectrales de campo simultáneas al paso del satélite. El objetivo de este trabajo es determinar y validar el protocolo óptimo para medir en el campo espectros de reflectividad (del visible al infrarrojo cercano) y emisividad (en el infrarrojo térmico) de muestras con características espectrales singulares (calcita, cuarzo, yeso y sal). Los espectrorradiómetros analizados son el Ocean Optics USB2000+ (200-1100 nm) y e

Foaming of Polycaprolactone and Its Impregnation with Quercetin Using Supercritical CO2

Foamed polycaprolactone impregnated with quercetin was carried out with a batch foaming technique using supercritical CO2. The experimental design was developed to study the influence of pressure (15–30 MPa), temperature (308–333 K), and depressurization rate (0.1–20) on the foam structure, melting temperature, and release tests of composites. The characterization of the experiments was carried out using scanning electron microscopy, X-ray di ractometer, and di erential scanning calorimetry techniques. It was observed that the porosity created in the polymer had a heterogeneous structure, as well as the impregnation of the quercetin during the process. On the other hand, controlled release tests showed a significant delay in the release of quercetin compared to commercial quercetin

Deposition of CAP/Antioxidants Systems on Silica Particles Using the Supercritical Antisolvent Process

Supercritical carbon dioxide has been used to deposit co-precipitates of natural antioxidants with a polymer onto silica microparticles. The supercritical antisolvent process (SAS) was carried out with the antioxidants by introducing the silica microparticles into the precipitator vessel. Two di erent configurations were employed to pump the solution. In one configuration, the antioxidant and the polymer were dissolved and injected together through a nozzle. In the second configuration, the antioxidant and the polymer were dissolved in di erent solutions and sprayed through di erent nozzles. The use of operating conditions significantly above the critical point (180 bar and 323 K) led to the formation of composites made up of co-precipitates and silica. Delivery profiles showed that the presence of the polymer and the silica delayed release of the antioxidant into gastric media, thus protecting it and allowing its full delivery to the intestinal fluids to improve the e ectiveness of the antioxidant

Precipitation of powerful antioxidant nanoparticles from orange leaves by means of supercritical CO2

Se ha llevado a cabo un amplio estudio en el que se ha utilizado CO2 como disolvente y antisolvente para apreciar las hojas de naranjo como fuente de potentes nanopartículas antioxidantes. En el primer caso, las hojas se procesaron directamente para obtener partículas del rango nanométrico o micrométrico. En el segundo caso, se realizó una extracción etanólica convencional de las hojas de naranjo antes de someterlas a condiciones supercríticas para extraer las partículas con alta actividad antioxidante. Así, se estudiaron diferentes niveles de presión (80-300 bar), temperatura (40-100 °C) y concentración de extracto líquido (10-42 mg/mL). Cuando se utilizó CO2 como disolvente, se precipitaron aglomerados con baja actividad antioxidante. Se requirieron mayores niveles de presión y temperatura para obtener la precipitación del polvo. Sin embargo, cuando se utilizó CO2 como antisolvente, precipitaron polifenoles con alta actividad antioxidante (3-4,65) en el rango nanométrico (60-73 nm). La eficacia del proceso se determinó por la relación de concentración de compuestos antioxidantes del extracto. La presión del recipiente fue la variable que más influyó en el tamaño de las partículas y la actividad antioxidante. El menor tamaño de partícula y el mayor rendimiento del precipitado se obtuvieron a alta presión y baja temperatura.An extensive study has been carried out where CO2 has been used both as solvent and antisolvent to appreciate orange leaves as a source of powerful antioxidant nanoparticles. In the former case, the leaves were processed directly to yield particles in the nano or micrometer range. In the latter case, a conventional ethanolic extraction of orange leaves was carried out before the orange leaves were subjected to supercritical conditions to extract the particles with high antioxidant activity. Thus, different pressure (80-300 bar), temperature (40-100 °C) and liquid extract concentration (10-42 mg/mL) levels were studied. When CO2 was used as the solvent, agglomerates with low antioxidant activity precipitated. Higher pressure and temperature levels were required to obtain powder precipitation. However, when CO2 was used as the antisolvent, polyphenols with high antioxidant activity (3-4.65) in the nanometer range (60-73 nm) would precipitate. The efficiency of the process was determined by the antioxidant compound concentration ratio of the extract. Vessel pressure was the main influencing variable with regards to impact on particle size and antioxidant activity. The smallest particle size and greatest precipitate yields were obtained at high pressure and low temperature

Co-precipitation of grape residue extract using sub- and supercritical CO2 technology

a micronización y secado de extractos líquidos crudos por procesos verdes no térmicos es muy interesante cuando se desea reducir el espacio de almacenamiento, los costos de transporte y concentrar y encapsular compuestos de interés. En este trabajo, se estudió la co-precipitación de extracto de etanol a partir de residuos de uva con un polímero biodegradable y biocompatible, polivinilpirrolidona (PVP), utilizando tecnología de CO2 subcrítica y supercrítica, es decir, en el presente trabajo mediante un antisolutorio presurizado (PAS). Se utilizó un diseño factorial completo (3 x 3) para evaluar el efecto de la presión (90, 130 y 170 bar) y la temperatura (20, 32 y 45 ºC) sobre el proceso PAS y las características de las partículas formadas por él. Presiones más altas (170 bar) y temperaturas (45 ºC) resultaron significativamente en condiciones operativas con con mejores rendimientos de precipitación global y en la producción de partículas con un diámetro medio más bajo y un contenido residual de etanol. Las partículas tenían una forma casi esférica con tamaños de 1,3 a 4,1 µm. La interacción de baja temperatura (20 ºC) con alta presión (170 bar) llevó a la producción de polvos secos más concentrados en antocianinas y compuestos fenólicos totales, y en procesos con mayores rendimientos de co-precipitación de estos bioactivos. Aunque los factores en estudio no tienen un efecto significativo sobre la capacidad antioxidante (CA) de las partículas, se observó que la CA se correlacionó fuertemente con el contenido de antocianinas y fenoles totales, demostrando así que el poder antioxidante del PAS co-precipitados del residuo de uva que era debido a la presencia de fenólico, principalmente antocianinas. Trece antocianinas fueron identificadas y cuantificadas en el extracto original y micropartículas co-precipitadas por UHPLC, siendo trans-malvidina-3-O-(6"-cumaril)-glucósido, malvidina-3-O-glucósido y malvidina-3-(6"-acetil)-glucósido las principales antocianinas. A partir de los estudios de liberación in vitro, se observó que las micropartículas, producidas en las mejores condiciones experimentales, liberaban compuestos fenólicos más lentamente en fluidos gastrointestinales simulados que el extracto precipitado sin polímero. Las antocianinas totales se detectaron parcialmente en el líquido intestinal, mientras que en el líquido gástrico se liberaron al 100% en un corto período. Al final, se puede concluir que la técnica del PAS, cuando se realiza principalmente en condiciones supercríticas, es un proceso muy prometedor para co-precipitación, concentración y fraccionamiento simultáneo de compuestos polares sensibles, como antocianinas, presentes en extractos de plantas crudas. Además, conduce a la producción de sistemas de administración bioactivos con mejores tasas de disolución en fluidos gastrointestinales simuladosThe micronization and drying of crude liquid extracts by green non-thermal processes is very interesting when it is desired to reduce storage space, transport costs, and to concentrate and encapsulate compounds of interest. In this work, the co-precipitation of ethanol extract from grape residue with a biodegradable and biocompatible polymer, polyvinylpyrrolidone (PVP), was studied using sub- and supercritical CO2 technology, namely in the present work by pressurized anti-solvent (PAS). A full factorial design (3 x 3) was used to evaluate the effect of pressure (90, 130 and 170 bar) and temperature (20, 32 and 45 ºC) on the PAS process and the characteristics of the particles formed by it. Higher pressures (170 bar) and temperatures (45 ºC) significantly resulted in operational conditions with better global precipitation yields and in the production of particles with a lower average diameter and residual ethanol content. The particles had a quasi-spherical shape with sizes from 1.3 to 4.1 µm. The interaction of low temperature (20 ºC) with high pressure (170 bar) led to the production of dry powders more concentrated in anthocyanins and total phenolic compounds, and in processes with higher co-precipitation yields of these bioactives. Although the factors under study do not have a significant effect on the antioxidant capacity (AC) of the particles, it was noted that AC correlated strongly with the content of anthocyanins and total phenolics, thus demonstrating that the antioxidant power of the PAS co-precipitates of the grape residue it was due to the presence of phenolic compounds, mainly anthocyanins. Thirteen anthocyanins were identified and quantified in the original extract and co-precipitated microparticles by UHPLC, being trans-malvidin-3-O-(6"-cumaryl)-glucoside, malvidin-3-O-glucoside and malvidin-3-(6"-acetyl)-glucoside the major anthocyanins. From the in vitro release studies, it was observed that the microparticles, produced from the best experimental conditions, released more slowly phenolic compounds in simulated gastrointestinal fluids than the precipitated extract without polymer. Total anthocyanins were partially detected in the intestinal fluid, while in the gastric fluid were 100% released in a short period. In the end, it can be concluded that the PAS technique, when performed mainly in supercritical conditions, is a very promising process for co-precipitating, concentrating and simultaneously fractionating sensitive polar compounds, such as anthocyanins, present in crude plant extracts. Furthermore, it leads to the production bioactive delivery systems with better dissolution rates in simulated gastrointestinal fluids

Espectroradiometría de campo del visible al infrarrojo térmico de muestras con características espectrales singulares

La calibración de sensores a bordo de satélite requiere medidas espectrales de campo simultáneas al paso del satélite. El objetivo de este trabajo es determinar y validar el protocolo óptimo para medir en el campo espectros de reflectividad (del visible al infrarrojo cercano) y emisividad (en el infrarrojo térmico) de muestras con características espectrales singulares (calcita, cuarzo, yeso y sal). Los espectrorradiómetros analizados son el Ocean Optics USB2000+ (200-1100 nm) y e

Análisis dendroclimático de Pinus pseudostrobus y Pinus devoniana en los municipios de Áporo y Zitácuaro (Michoacán), Reserva de la Biósfera de la Mariposa Monarca

Se presenta el primer estudio sobre dendroclimatología de Pinus pseudostrobus y Pinus devoniana en el estado de Michoacán (México). El estudio se realizó en los municipios de Áporo y Zitácuaro de la Reserva de la Biosfera de la Mariposa Monarca (RBMM). Se colectaron cien testimonios de madera tomados a 1.3 m de altura del árbol, repartidas en los ejidos del Rincón del Soto y Arroyo Seco (Áporo), y en el ejido de San Juan de Zitácuaro. Las series dendrocronológicas en Áporo para la especie Pinus pseudostrobus se extendió a 62 años (1949-2010) y para Pinus devoniana 86 años (1925-2010); y las series en Zitácuaro para Pinus pseudostrobus y Pinus devoniana se extendieron a 47 años (1964-2010) y 44 años (1967-2010), respectivamente. Con el objetivo de establecer los parámetros climáticos que controlan el crecimiento radial de las especies estudiadas, se encontró que la serie potencial para hacer estudios dendrocronológicos es la de Pinus pseudostrobus en Zitácuaro. El valor de la serie de Pinus devoniana se encuentra ligeramente por debajo (0.81) del valor de aceptación (0.85). Las series de crecimiento radial se compararon con datos climáticos de precipitación mensual, temperatura media mensual, temperatura máxima y mínima medias mensuales, de estaciones meteorológicas cercanas al área en estudio. Las funciones de correlación indican que la anchura de los anillos de ambas especies está regulada significativamente por la precipitación y la temperatura máxima media en Zitácuaro. Ambas especies reaccionan semejantemente con el clima, pero con algunas diferencias. Para Pinus pseudostrobus hay una relación positiva del crecimiento radial con el aumento de precipitación en el mes de abril del año actual; y hay una relación negativa del crecimiento radial con el aumento de temperatura máxima media en el mes de agosto del año previo. Para Pinus devoniana hay una relación negativa del crecimiento radial con el aumento de precipitación en el mes de noviembre del año previo, y una relación positiva en febrero del año actual; y hay una relación negativa del crecimiento radial con el aumento de temperature máxima media en los meses de julio y agosto del año previo.This article presents the first study on dendroclimatology of Pinus pseudostrobus and Pinus devoniana in the state of Michoacán (Mexico), specifically in the municipalities of Áporo and Zitácuaro, both municipalities within the Monarch Butterfly Biosphere Reserve (MBBR). The sampling in Áporo, northwest of the MBBR, was held in Los Ejidos del Rincón del Soto and Arroyo Seco, in Sierra Chincúa (May 2011). In Zitácuaro, southwest of the reserve, a sampling was performed in the Ejido de San Juan de Zitácuaro, in the area of Ocotal and Palma, and Meso Sedano (June 2011). There were a total of 38 Pinus pseudostrobus and 12 Pinus devoniana sampled in both areas of the study and distributed in 28 trees in the municipality of Áporo and 22 in Zitácuaro. Two samples per tree were taken at 1.3 m height, resulting in a total of 100 tree cores. The dendrochronological series in Áporo for the species Pinus pseudostrobus were extended to 62 years (1949- 2010) and for Pinus devoniana 86 years (1925-2010); and the series in Zitácuaro for Pinus pseudostrobus and Pinus devoniana were extended to 47 years (1964-2010) and 44 years (1967-2010), respectively. The ring chronologies were validated using the program COFECHA, which calculates the cross correlations between individual series of the tree-growth, five series were eliminated due to very low or negative correlations. The climate data from Zitácuaro were obtained from two weather stations located in the same municipality. And, in the case of Áporo, the data was obtained from stations located in Senguio. The growth rates related to the climate were obtained by removing the growth trend of each tree due to the age, size and other factors such as the competition, using the program ARSTAN. The following statistics were used to evaluate the quality of the residual chronologies and to determine the potential dendrochronology of species for the different populations: the average correlation between series (Rbar), the signal expressed by the population (EPS), medium sensitivity (MS), and the auto-correlation of first order (AC). To analyze the correlation and the function of response between the radial growth and climate variables, the waste medium chronologies of the growth rates for both species, and the records of temperature and precipitation, were evaluated through the Dendroclim2002, which determines the period of the year that most influences the radial growth of Pinus in relation to monthly climate data. In order to establish the climatic parameters that control the radial growth of the studied species, the Pinus pseudostrobus was found to be the potential series to perform the dendrochronological study in Zitácuaro. The value of the series of Pinus devoniana is slightly lower (0.81) than the acceptance value (0.85). The series of radial growth were compared with the climate records of monthly precipitation, average monthly temperature, and average maximum and minimum monthly temperatures. Correlation functions indicate that the width of the rings of both species is regulated significantly by rainfall and the average maximum temperature in Zitácuaro. Both species react similarly to the climate, but with some differences. There is a positive relationship between radial growth of Pinus pseudostrobus and the increasing rainfall in April of the current year; and there is a negative relationship between radial growth and the increasing average maximum temperature in August the previous year. For Pinus devoniana there is a negative relationship between radial growth and the increasing rainfall in November of the previous year, and a positive relationship in February of the current year; and there is a negative relationship between radial growth and the increasing average of maximum temperature in July and August last year