In vitro cytotoxicity assessment of monocationic and dicationic pyridinium-based ionic liquids on HeLa, MCF-7, BGM and EA.hy926 cell lines

Dicationic ionic liquids (ILs) generally possess higher thermal and electrochemical stability than the analogous monocationic ILs, which makes them more suitable for high-temperature applications as solvents for organic reactions, lubricants or stationary phase in gas chromatography. However, knowledge on dicationic IL cytotoxicity is still scarce. Here we explore the cytotoxicity of twelve mono- and dicationic pyridinium-based ILs on HeLa, MCF-7, BGM and EA.hy926 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle arrest assays, apoptosis experiments and orange staining were carried out. The results showed that dicationic ILs are generally less cytotoxic than their monocationic counterparts. In monocationic ILs, cytotoxicity was stronger when they contain long alkyl chains, because of their higher lipophilicity. However, the full effect of the length of the linkage alkyl chain of dicationic ILs on cytotoxicity is not clear probably because the chain is “trapped” between both cationic moieties. IL cytotoxicity is highly dependent on the cell type, and HeLa cells exposed to [C12Pyr]Br die via apoptosis. The present study increases our knowledge of IL cytotoxicity on human and monkey cells and clarifies the cell death mechanism. The results suggest that dicationic ILs offer the potential to replace some monocationic ILs because of their lower cytotoxicity.This work was partially supported by the European Commission (FEDER/ERDF) and the Spanish MINECO (Ref. CTQ2017-87708-R) and the research support programme of the Seneca Foundation of Science and Technology of Murcia, Spain (Ref. 20977/PI/18). M. G. Montalbán acknowledges support from MINECO (Juan de la Cierva-Formación contract, Ref. FJCI-2016-28081), P. Licence acknowledges EPSRC and BBSRC for support (EP/S005080/1, EP/P013341/1, BB/L013940/1)

Nanoparticles as Drug Delivery Systems

This chapter presents a review on the design of nanoparticles which have been proposed as drug delivery systems in biomedicine. It will begin with a brief historical review of nanotechnology including the most common types of nanoparticles (metal nanoparticles, liposomes, nanocrystals and polymeric nanoparticles) and their advantages as drug delivery systems. These advantages include the mechanism of increased penetration and retention, the transport of insoluble drugs and the controlled release. Next, the nanoparticle design principles and the routes of administration of nanoparticles (parental, oral, pulmonary and transdermal) are discussed. Different routes of elimination of nanoparticles (renal and hepatic) are also analyzed

Silk Fibroin Nanoparticles: Synthesis and Applications as Drug Nanocarriers

The use of nanoparticles in biomedical fields is a very promising scientific area and has aroused the interest of researchers in the search for new biodegradable, biocompatible and non-toxic materials. This chapter is based on the features of the biopolymer silk fibroin and its applications in nanomedicine. Silk fibroin, obtained from the Bombyx mori silkworm, is a natural polymeric biomaterial whose main features are its amphiphilic chemistry, biocompatibility, biodegradability, excellent mechanical properties in various material formats, and processing flexibility. All of these properties make silk fibroin a useful candidate to act as nanocarrier. In this chapter, the structure of silk fibroin, its biocompatibility and degradability are reviewed. In addition, an intensive review on the silk fibroin nanoparticle synthesis methods is also presented. Finally, the application of the silk fibroin nanoparticles for drug delivery acting as nanocarriers is detailed

Effect of Degumming in the Characteristics of Silk Fibroin Nanoparticles

Several studies have stated that the process used for sericin removal, or degumming, from silk cocoons has a strong impact on the silk fibroin integrity and consequently in their mechanical or biochemical properties after processing it into several biomaterials (e.g., fibers, films or scaffolds) but still, there is a lack of information of the impact on the features of silk nanoparticles. In this work, silk cocoons were degummed following four standard methods: autoclaving, short alkaline (Na2CO3) boiling, long alkaline (Na2CO3) boiling, and ultrasounds. The resultant silk fibroin fibers were dissolved in the ionic liquid 1-ethyl-3-methylimidazolium acetate and used for nanoparticle synthesis by rapid desolvation in polar organic solvents. The relative efficiencies of the degumming processes and the integrity of the resulting fibroin fibers obtained were analyzed by mass loss, optical microscopy, thermogravimetric analysis, infrared spectroscopy, and SDS-PAGE. Particle sizes and morphology were analyzed by Dynamic Light Scattering and Field Emission Scanning Electronic Microscopy. The results showed that the different treatments had a remarkable impact on the integrity of the silk fibroin chains, as confirmed by gel electrophoresis, which can be correlated with particle mean size and size distribution changes. The smallest nanoparticles (156 ± 3 nm) and the most negative Z potential (−30.2 ± 1.8 mV) were obtained with the combination of long treatment (2 h) of boiling in alkaline solution (Na2CO3 0.02 eq/L). The study confirms that parameters of the process, such as the composition of the solution and time of the degumming step, must be controlled in order to reach an optimum reproducibility of the nanoparticle production.This work has been partially supported (80%) by the European Commission ERDF/FEDER Operational Programme 'Murcia' CCI N° 2007ES161PO001 (Project No. 14-20/20), and the Spanish MINECO (Ref. CTQ2017-87708-R) and the programme of support to the research of the Seneca Foundation of Science and Technology of Murcia, Spain (Ref. 20977/PI/18). A.A.L.-P.’s research contract was partially supported (80%) by the ERDF/FEDER Operational Programme 'Murcia' CCI N° 2007ES161PO001 (Project No. 14-20/20),. M.G. Montalbán’s research contract is funded by the Spanish MINECO (Juan de la Cierva-Formación contract, Ref. FJCI-2016-28081). S.D.A.-C.’s research contract is funded by the program INIA-CCAA (DOC INIA 2015), announced by the National Institute for Agricultural and Food Research and Technology (INIA) and supported by the Spanish State Research Agency (AEI) under the Spanish Ministry of Economy, Industry and Competitiveness

Biopolymeric Nanoparticle Synthesis in Ionic Liquids

Recently, much research has focused on the use of biopolymers, which are regarded as biodegradable, natural, and environmentally friendly materials. In this context, biopolymeric nanoparticles have attracted great attention in the last few years due to their multiple applications especially in the field of biomedicine. Ionic liquids have emerged as promising solvents for use in a wide variety of chemical and biochemical processes for their extraordinary properties, which include negligible vapor pressure, high thermal and chemical stability, lower toxicity than conventional organic solvents, and the possibility of tuning their physical–chemical properties by choosing the appropriate cation and anion. We here review the published works concerning the synthesis of biopolymeric nanoparticles using ionic liquids, such as trimethylsilyl cellulose or silk fibroin. We also mention our recent studies describing how high-power ultrasounds are capable of enhancing the dissolution process of silk proteins in ionic liquids and how silk fibroin nanoparticles can be directly obtained from the silk fibroin/ionic liquid solution by rapid desolvation in polar organic solvents. As an example, their potential biomedical application of curcumin-loaded silk fibroin nanoparticles for cancer therapy is also discussed

Revealing the Influence of the Degumming Process in the Properties of Silk Fibroin Nanoparticles

Several studies have stated that the process used for sericin removal, or degumming, from silk cocoons has a strong impact in the silk fibroin integrity and consequently in their mechanical or biochemical properties after processing it into several biomaterials (e.g. fibers, films or scaffolds) but still, there is a lack of information of the impact on the features of silk nanoparticles. In this work, silk cocoons were degummed following four standard methods: autoclaving, short alkaline (Na2CO3) boiling, long alkaline (Na2CO3) boiling and ultrasounds. The resultant silk fibroin fibers were dissolved in the ionic liquid 1-ethyl-3-methylimidazolium acetate and used for nanoparticle synthesis by rapid desolvation in polar organic solvents. The relative efficiencies of the degumming processes and the integrity of the resulting fibroin fibers obtained were analyzed by mass loss, optical microscopy, thermogravimetric analysis, infrared spectroscopy and SDS-PAGE. Particle sizes and morphology were analyzed by Dynamic Light Scattering and Field Emission Scanning Electronic Microscopy. The results showed that the different treatments had a remarkable impact on the integrity of the silk fibroin chains, as confirmed by gel electrophoresis, which can be correlated with particle mean size and size distribution changes. The smallest nanoparticles (156 ± 3 nm) and the most negative Z potential (−30.2 ± 1.8 mV) were obtained with the combination of long treatment (2 h) of boiling in alkaline solution (Na2CO3 0.02 eq/L). The study confirms that parameters of the process, such as composition of the solution and time of the degumming step, must be controlled in order to reach an optimum reproducibility of the nanoparticle production.This work has been partially supported (80%) from the European Commission ERDF/FEDER Operational Programme 'Murcia' CCI N° 2007ES161PO001 (Project No. 14-20/20), and the Spanish MINECO (Ref. CTQ2017-87708-R) and the programme of support to the research of the Seneca Foundation of Science and Technology of Murcia, Spain (Ref. 20977/PI/18).A.A. Lozano-Pérez’s research contract was partially supported (80%) by the ERDF/FEDER Operational Programme 'Murcia' CCI N° 2007ES161PO001 (Project No. 14-20/20),. M.G. Montalbán’s research contract is funded by the Spanish MINECO (Juan de la Cierva-Formación contract, Ref. FJCI-2016-28081). S.D. Aznar-Cervantes’s research contract is funded by the program INIA-CCAA (DOC INIA 2015), announced by the National Institute for Agricultural and Food Research and Technology (INIA) and supported by The Spanish State Research Agency (AEI) under the Spanish Ministry of Economy, Industry and Competitiveness

Direct Quantification of Drug Loading Content in Polymeric Nanoparticles by Infrared Spectroscopy

Nanotechnology has enabled the development of novel therapeutic strategies such as targeted nanodrug delivery systems, control and stimulus-responsive release mechanisms, and the production of theranostic agents. As a prerequisite for the use of nanoparticles as drug delivery systems, the amount of loaded drug must be precisely quantified, a task for which two approaches are currently used. However, both approaches suffer from the inefficiencies of drug extraction and of the solid-liquid separation process, as well as from dilution errors. This work describes a new, reliable, and simple method for direct drug quantification in polymeric nanoparticles using attenuated total reflection Fourier transform infrared spectroscopy, which can be adapted for a wide variety of drug delivery systems. Silk fibroin nanoparticles and naringenin were used as model polymeric nanoparticle carrier and drug, respectively. The specificity, linearity, detection limit, precision, and accuracy of the spectroscopic approach were determined in order to validate the method. A good linear relation was observed within 0.00 to 7.89% of naringenin relative mass with an R2 of 0.973. The accuracy was determined by the spike and recovery method. The results showed an average 104% recovery. The limit of detection and limit of quantification of the drug loading content were determined to be 0.3 and 1.0%, respectively. The method’s robustness is demonstrated by the notable similarities between the calibrations carried out using two different equipment setups at two different institutions

Síntesis y caracterización de nanopartículas de fibroína de seda para el transporte de fármacos

La academia y la industria han conseguido avances extraordinarios en una gran variedad de áreas debido al desarrollo de la nanotecnología y el control de estructuras a niveles nanoscópicos. Particularmente, en el campo de la medicina, la nanotecnología tiene el potencial de generar un impacto significativo en la salud humana pudiendo mejorar el diagnóstico, la prevención y el tratamiento de enfermedades. En este campo, la nanotecnología busca encapsular fármacos y/o compuestos trazadores en nanopartículas con el fin de aumentar la eficacia de los mismos al permitir un suministro directo en tejidos diana; al mismo tiempo que reducen su toxicidad evitando la acumulación y los consiguientes efectos secundarios en tejidos sanos. La encapsulación de fármacos también permite su liberación controlada pudiendo así evitar máximos de concentraciones elevadamente dañinas o subterapéuticas. Sumado a esto, las nanopartículas han demostrado ser de gran valor en el transporte de fármacos con baja solubilidad en agua; el cual resulta ser el principal problema a la hora de introducir nuevos medicamentos al mercado debido a que este hecho limita su biodisponibilidad en el organismo. La fibroína de seda del gusano de seda de la especie Bombyx mori es un polímero natural, proteico, que presenta una interesante combinación de propiedades mecánicas, tales como flexibilidad y resistencia que aún son difíciles de conseguir con polímeros sintéticos. Además, la fibroína es biodegradable y biocompatible, lo que la convierte en un excelente material para la producción de nanopartículas para la administración de fármacos. Las nanopartículas de fibroína de seda son capaces de cargar una amplia variedad de compuestos terapéuticos y diversas macromoléculas, penetrar membranas biológicas y ser modificadas químicamente para ampliar su funcionalidad. Si bien la notable resistencia mecánica de la fibroína es una de las propiedades atractivas del biomaterial, el proceso de síntesis de nanopartículas se encuentra obstaculizado por su elevada estabilidad. Un método reciente, desarrollado por el Grupo de Investigación en el que se ha realizado la presente tesis doctoral, basado en la utilización de líquidos iónicos para disolver la fibroína nativa, ha permitido la producción de nanopartículas en un proceso fácil y escalable a la industria. El proceso de síntesis de nanopartículas de fibroína de seda consta de varias etapas. En primer lugar, se lleva a cabo la purificación de la fibroína, mediante la eliminación de la sericina (método conocido como desgomado). En segundo lugar, la fibroína debe ser disuelta en sus unidades monoméricas y, por último, ésta es regenerada en forma de nanopartículas. Cada uno de estos procesos puede tener efectos significativos en la estructura secundaría de la proteína y dadas las implicaciones que ésta tiene en la resistencia, degradabilidad y biocompatibilidad del producto final, su estudio resulta indispensable. Por otro lado, para la correcta administración de dosis terapéuticas, los fármacos cargados en las nanopartículas deben ser cuantificados con precisión. Hasta la fecha, dos enfoques son utilizados principalmente para esta tarea. Los cuales están basados en una medición directa o indirecta del contenido de carga de fármaco en la nanopartícula. Sin embargo, ambos adolecen de las ineficiencias de la extracción del fármaco y del proceso de separación sólido-líquido, así como de errores de dilución. Dadas las consideraciones anteriores, esta Tesis doctoral se centra en el estudio de las nanopartículas de fibroína de seda como nanotransportadores de fármacos, su proceso de síntesis utilizando líquidos iónicos, la caracterización de la estructura secundaría de la proteína durante todo el proceso de síntesis y la cuantificación de la cantidad de fármaco cargado en las nanopartículas.The academy and industry have made extraordinary advances in a wide variety of areas due to the development of nanotechnology and the control of structures at the nanoscopic levels. Particularly in the field of medicine, nanotechnology has the potential to generate a significant impact on human health, being able to improve the diagnosis, prevention and treatment of diseases. In this field, nanotechnology seeks to encapsulate drugs and / or tracer compounds in nanoparticles to increase their efficiency by allowing direct delivery to target tissues; while they reduce their toxicity avoiding accumulation and the consequent side effects in healthy tissues. The encapsulation of drugs also allows their controlled release, thus avoiding maximum levels of highly harmful or subtherapeutic concentrations. Moreover, nanoparticles are of great value in the transport of drugs with low solubility in water; which turns out to be the major problem when introducing new drugs to the market because it limits their bioavailability in the body. The silk fibroin of the Bombyx mori species silkworm is a natural, protein polymer that presents an interesting combination of mechanical properties, such as flexibility and resistance which are still difficult to achieve with synthetic polymers. Furthermore, fibroin is biodegradable and biocompatible, which makes it an excellent material for the production of nanoparticles for drug delivery. Silk fibroin nanoparticles are capable of loading a wide variety of therapeutic compounds and various macromolecules, penetrate biological membranes, and be chemically modified to extend their functionality. Although the remarkable mechanical resistance of fibroin is one of the attractive properties of the biomaterial, the nanoparticle synthesis process is hampered by its high stability, due to the high number of hydrogen bonds in its secondary structure, mostly in the form of antiparallel β sheet. A recent method, developed by the Research Group in which this doctoral thesis has been carried out, based on the use of ionic liquids to dissolve native fibroin, has allowed the production of nanoparticles in an easy and scalable process for industry. The silk fibroin nanoparticle synthesis process comprises several stages. First, the fibroin is purified by removing the sericin (a method known as degumming). Second, fibroin must be dissolved in its monomeric units and, subsequently, regenerated into nanoparticles. Each of these steps can have significant effects on the secondary structure of the protein and given the implications it has on the resistance, degradability and biocompatibility of the final product, their study is essential. On the other hand, for the correct administration of therapeutic doses, the drugs loaded in the nanoparticles must be precisely quantified. To date, two approaches are mainly used for this task. They are based on a direct and indirect measurement of the drug loading content in the nanoparticle. However, both suffer from inefficiencies in drug extraction and solid-liquid separation process, as well as dilution errors. Given the above considerations, this Doctoral Thesis focuses on the study of silk fibroin nanoparticles as drug nanocarriers, their synthesis process using ionic liquids, the characterization of the secondary structure of the protein during the entire synthesis process and the quantification of the amount of drug in the nanoparticles

Preparación y caracterización de nanofluidos de alúmina con aplicaciones térmicas

Los nanofluidos son un tipo de fluidos cuya fase dispersa está constituida por partículas de tamaño nanométrico. Su interés viene dado por el hecho de que presentan una mejora en las magnitudes térmicas y eléctricas respecto a los fluidos base, empleándose en biomedicina, el sector automovilístico o en dispositivos de energía solar térmica. Por este motivo, nos propusimos obtener y determinar las propiedades de un nanofluido que fuera apropiado para utilizar en un colector solar plano como fluido caloportador, proporcionando así una mejora en su rendimiento. En este trabajo se han preparado nanofluidos de alúmina, Al2O3, de concentración en volumen 0,62 % en agua milliQ dispersos mediante la técnica de sonicación. Este tipo de nanopartículas son de las más recurridas en experimentaciones de energía solar térmica por su mejora en el rendimiento de las instalaciones a costa de su baja inversión. Una vez preparados los nanofluidos, se estudiaron ciertos parámetros tanto miscroscópicos como macroscópicos como el diámetro hidrodinámico, el potencial zeta, el diámetro de partícula y la conductividad térmica. Los estudios realizados mediante observación al microscopio indican que el diámetro de partícula es de 13 nm, similar al que indica el fabricante, mientras que los diámetros hidrodinámicos obtenidos por dispersión de luz dinámica son 10 veces mayores. El potencial zeta de las nanopartículas es aproximadamente 60 mV, valor que supone que la estabilidad de la dispersión es correcta. Por último, se ha estimado la mejora en la conductividad térmica para la concentración de 0,62 % obteniendo un valor de knf/kbf de 1,018. No se ha apreciado una diferencia significativa en los tamaños de partícula comparando los experimentos realizados a diferentes tiempos y amplitudes de sonicación. Para más índole, aumentar la agresividad del tratamiento supone el desprendimiento de partículas de la punta del sonicador, alterando las muestras. En cuanto a la estabilidad de la dispersión, tras un periodo de 1 mes, se ha comprobado que las nanopartículas no se han agregado

Density, Refractive Index and Volumetric Properties of Water–Ionic Liquid Binary Systems with Imidazolium-Based Cations and Tetrafluoroborate, Triflate and Octylsulfate Anions at T = 293 to 343 K and p = 0.1 MPa

The density and refractive index of ionic liquids (ILs) + water binary mixtures were determined as a function of temperature (from 293.15 to 343.15 K) at atmospheric pressure over the whole composition range in which the mixtures were miscible. To carry out a systematic study, all of the ILs selected are imidazolium-based ILs with a different number of carbons in the alkyl chain of the cation and also different anions (tetrafluoroborate, triflate, and octylsulfate). Specifically, the studied ILs were 1-ethyl-3-methylimidazolium tetrafluoroborate [emim][BF4], 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF4], 1-hexyl-3-methylimidazolium tetrafluoroborate [hmim][BF4], 1-methyl-3-octylimidazolium tetrafluoroborate [omim][BF4], 1-ethyl-3-methylimidazolium triflate [emim][TfO], 1-butyl-3-methylimidazolium triflate [bmim][TfO], and 1-butyl-3-methylimidazolium octylsulfate [bmim][OcSO4]. The excess molar volumes and the deviation in the molar refraction of the binary mixtures were calculated for a better understanding of the interactions that take place between the components and were successfully correlated by the Redlich–Kister empirical correlations. The Bahe–Varela model, which has a more physical meaning, was also used to successfully correlate the excess molar volume values. Volumetric properties, such as apparent molar volumes, partial molar volumes, isobaric thermal expansion coefficients, partial molar volumes at infinite dilution and excess partial molar volumes at infinite dilution were also calculated in order to obtain information about the influence of composition and temperature on the thermodynamic behavior of the selected ILs and water in the mixture. The results are discussed in order to understand the formation of hydrogen bonds between components of the mixture and the possible packing effects that take place in the mixing process. The density and refractive index experimental data were correlated by the Lorentz–Lorenz, Wiener, Dale–Gladstone, and Eykman equations to determine the relationship between both parameters, and good agreement between the experimental and calculated refractive index values was obtained.This work has been partially supported by the European Commission (FEDER/ERDF), the Spanish MINECO (ref. CTQ2014-57467-R and ref. CTQ2017-87708-R), and the programme of support for the research of the Seneca Foundation of Science and Technology of Murcia, Spain (ref. 19499/PI/14)