https://hdl.handle.net/10481/14641 2026-04-05T09:18:11Z https://hdl.handle.net/10481/111526 Using AFM to probe the complexation of DNA with anionic lipids mediated by Ca2+: the role of surface pressure Luque Caballero, Germán; Martín Molina, Alberto; Sánchez Treviño, Alda Yadira; Rodríguez Valverde, Miguel Ángel; Cabrerizo Vílchez, Miguel Ángel; Maldonado Valderrama, Julia Complexation of DNA with lipids is currently being developed as an alternative to classical vectors based on viruses. Most of the research to date focuses on cationic lipids owing to their spontaneous complexation with DNA. Nonetheless, recent investigations have revealed that cationic lipids induce a large number of adverse effects on DNA delivery. Precisely, the lower cytotoxicity of anionic lipids accounts for their use as a promising alternative. However, the complexation of DNA with anionic lipids (mediated by cations) is still in early stages and is not yet well understood. In order to explore the molecular mechanisms underlying the complexation of anionic lipids and DNA we proposed a combined methodology based on the surface pressure–area isotherms, Gibbs elasticity and Atomic Force Microscopy (AFM). These techniques allow elucidation of the role of the surface pressure in the complexation and visualization of the interfacial aggregates for the first time. We demonstrate that the DNA complexes with negatively charged model monolayers (DPPC/DPPS 4 : 1) only in the presence of Ca2+, but is expelled at very high surface pressures. Also, according to the Gibbs elasticity plot, the complexation of lipids and DNA implies a whole fluidisation of the monolayer and a completely different phase transition map in the presence of DNA and Ca2+. AFM imaging allows identification for the first time of specific morphologies associated with different packing densities. At low surface coverage, a branched net like structure is observed whereas at high surface pressure fibers formed of interfacial aggregates appear. In summary, Ca2+ mediates the interaction between DNA and negatively charged lipids and also the conformation of the ternary system depends on the surface pressure. Such observations are important new generic features of the interaction between DNA and anionic lipids.; La complejación de ADN con lípidos se está desarrollando actualmente como una alternativa a los vectores clásicos basados en virus. La mayoría de las investigaciones hasta la fecha se centran en lípidos catiónicos debido a su complejación espontánea con el ADN. Sin embargo, investigaciones recientes han revelado que los lípidos catiónicos producen un gran número de efectos secundarios. Precisamente, la menor citotoxicidad de los lípidos aniónicos avala su uso como alternativa prometedora. Sin embargo, el estudio de la complejación de ADN con lípidos aniónicos (mediada por cationes) aún se encuentra en su etapa inicial y no se ha comprendido en profundidad. Para explorar los mecanismos moleculares que subyacen a la complejación de lípidos aniónicos con ADN, proponemos una metodología experimetal que combina isotermas de presión superficial-área, elasticidad de Gibbs y Microscopía de Fuerzas Atómicas (AFM). Estas técnicas permiten dilucidar el papel de la presión superficial en la complejación y visualizar por primera vez los agregados interfaciales. Demostramos que el ADN se une a monocapas modelo cargadas negativamente (DPPC/DPPS 4:1) sólo en presencia de Ca²⁺, siendo expulsado a presiones superficiales muy altas. Además, según la gráfica de elasticidad de Gibbs, la complejación de lípidos y ADN implica una total fluidificación de la monocapa, con un diagrama de transición de fase completamente diferente en presencia de ADN y Ca²⁺. La obtención de imágenes por AFM permite identificar por primera vez las morfologías específicas para diferentes niveles de empaquetamiento superficial. A bajo recubrimiento superficial se observa una estructura de red ramificada, mientras que a alta presión superficial aparecen fibras formadas por agregados interfaciales. En resumen, el Ca²⁺ media la interacción entre el ADN y los lípidos cargados negativamente, dependiendo la configuración del sistema ternario de la presión superficial. Estas observaciones revelan nuevas e importantes características de la interacción entre el ADN y los lípidos aniónicos. https://hdl.handle.net/10481/111480 Adsorption of DNA onto anionic lipid surfaces Martín Molina, Alberto; Luque Caballero, Germán; Faraudo, Jordi; Quesada Pérez, Manuel; Maldonado Valderrama, Julia Currently self-assembled DNA delivery systems composed of DNA multivalent cations and anionic lipids are considered to be promising tools for gene therapy. These systems become an alternative to traditional cationic lipid–DNA complexes because of their low cytotoxicity lipids. However, currently these nonviral gene delivery methods exhibit low transfection efficiencies. This feature is in large part due to the poorly understood DNA complexation mechanisms at the molecular level. It is well-known that the adsorption of DNA onto like charged lipid surfaces requires the presence of multivalent cations that act as bridges between DNA and anionic lipids. Unfortunately, the molecular mechanisms behind such adsorption phenomenon still remain unclear. Accordingly a historical background of experimental evidence related to adsorption and complexation of DNA onto anionic lipid surfaces mediated by different multivalent cations is firstly reviewed. Next, recent experiments aimed to characterise the interfacial adsorption of DNA onto a model anionic phospholipid monolayer mediated by Ca2+ (including AFM images) are discussed. Afterwards, modelling studies of DNA adsorption onto charged surfaces are summarised before presenting preliminary results obtained from both CG and all-atomic MD computer simulations. Our results allow us to establish the optimal conditions for cation-mediated adsorption of DNA onto negatively charged surfaces. Moreover, atomistic simulations provide an excellent framework to understand the interaction between DNA and anionic lipids in the presence of divalent cations. Accordingly, our simulation results in conjunction go beyond the macroscopic picture in which DNA is stuck to anionic membranes by using multivalent cations that form glue layers between them. Structural aspects of the DNA adsorption and molecular binding between the different charged groups from DNA and lipids in the presence of divalent cations are reported in the last part of the study. Although this research work is far from biomedical applications, we truly believe that scientific advances in this line will assist, at least in part, in the rational design and development of optimal carrier systems for genes and applicable to other drugs.; Actualmente, los sistemas autoensamblados formados por ADN, cationes multivalentes y lípidos están considerados como herramientas prometedoras para la terapia genética. Estos sistemas suponen una alternativa a los complejos de ADN con lípidos catiónicos debido a su menor citotoxicidad. Sin embargo, estos vectores de transfección no víricos presentan bajas eficiencias de transfección. Esto se debe en gran parte a la poca información que tenemos sobre los mecanismos de esta complejación a nivel molecular. Es bien sabido que la complejación de ADN con superficies cargadas del mismo signo requiere la presencia de cationes multivalentes que actúan como puentes entre el ADN y los lípidos aniónicos. Desafortunadamente, los mecanismos moleculares que posibilitan esta interacción aún no están claros. En primer lugar, revisamos la historia de la evidencia experimental relacionada con la adsorción y complejación de ADN con superficies de lípidos aniónicas mediadas por diferentes cationes multivalentes. A continuación, discutimos experimentos recientes que caracterizan la adsorción interfacial de ADN sobre monocapas modelo con fosfolípidos aniónicos mediadas por Ca2+ (se incluyen imágenes de AFM). Después, resumimos los estudios teóricos de adsorción de ADN sobre superficies cargadas para después presentar los resultados preliminares de las simulaciones computacionales, distinguiendo por un lado los modelos de Grano Grueso (GG) y por otro los modelos atomísticos con el método de la Dinámica Molecular (DM). Nuestros resultados permiten establecer las condiciones óptimas para la adsorción de ADN sobre superficies con carga negativa mediadas por cationes. Además, las simulaciones atomísticas suponen una aproximación excelente para comprender todos los aspectos relacionados con la interacción entre el ADN y los lípidos aniónicos en presencia de cationes divalentes. Así, los resultados de nuestras simulaciones van más allá de la representación esquemática en la que el ADN se une a las membranas de lípidos aniónicos por medio de una capa de cationes que actúan como un pegamento. En la parte final del estudio describimos los aspectos estructurales involucrados en la adsorción de ADN en presencia de cationes de Ca2+, indicando la unión de los grupos cargados del ADN y de los lípidos. Aunque este trabajo de investigación se encuentra alejado de las aplicaciones biomédicas, creemos que los avances científicos en esta línea ayudarán, al menos en parte, al diseño y desarrollo de vectores genéticos y sistemas de transporte de otros fármacos. https://hdl.handle.net/10481/109479 Surface activity of protein extracts from seed oil by-products and wettability of developed bioplastics Mirpoor, Seyedeh Fatemeh; Ibáñez-Ibañéz, Pablo F.; Giosafatto, C. Valeria L.; del Castillo-Sanataella, Teresa; Rodriguez-Valverde, Miguel Angel; Maldonado-Valderrama, Julia Protein concentrates, extracted from the by-products of Hemp (Cannabis sativa), Cardoon (Cynara cardunculus), and Argan (Argania spinosa L.) oilseeds after oil extraction, were utilized to produce sustainable and eco-friendly protein-based bioplastics. The surface wettability and roughness of these bioplastics were investigated due to the pivotal role of these parameters in their application in food packaging sector. Advancing contact angle value revealed that Argan protein-based bioplastic is the most hydrophobic (θ > 90◦) while Cardoon protein-based film is the most hydrophilic (θ < 40◦). These results are then supported by the roughness of wet film surfaces and their swelling ratio, where Cardoon-based bioplastics and Argan-based bioplastics have also the lowest and the highest roughness average, respectively. The measured hydrophilicity of the produced bioplastics motivates a detailed investigation of the surface activity of protein concentrates used for film formation which can determine the packaging by coating. Adsorption/desorption kinetics, surface tension and dilatational rheology are reported at two different pHs values (10 and 12). The three protein concentrates are surface active and develop stable adsorbed layers at the air-water interface after 1 h. Cardoon proteins showed the same final surface tension (>45 mN m􀀀 1) at both pHs, while Argan and Hemp proteins displayed lower values than Cardoon, especially at pH 12 (<40 mN m􀀀 1), indicative of higher surface activity. Finally, Argan protein adsorbed more irreversibly, followed by Hemp protein while Cardoon protein provides the largest partial desorption from the surface. Combined analysis of the data features a correlation between the higher advancing contact angle of Hemp protein-based bioplastic and higher hydrophobicity, less swelling, faster adsorption kinetics, lower surface tension and more irreversible adsorption of protein. These apply for Hemp and Cardoon protein concentrates and their driven bioplastics while the presence of free oil in Argan protein concentrate and its bioplastic hinders the correlation between different phenomena. The new interconnections highlighted between film properties and surface adsorption could potentially be employed in food packaging and coating technology. https://hdl.handle.net/10481/102876 Interaction of DNA with likely-charged lipid monolayers: an experimental study Luque Caballero, Germán; Maldonado Valderrama, Julia; Quesada Pérez, Manuel; Martín Molina, Alberto Los lípidos aniónicos se están usando cada vez más como alternativa a los lípidos catiónicos para la producción de vectores genéticos sintéticos (lipoplejos). Esto se debe primordialmente a su menor toxicidad y biocompatibilidad. Sin embargo, los lipoplejos aniónicos requieren la presencia de cationes multivalentes para promover la atracción electrostática entre el ADN y las monocapas o bicapas aniónicas. En este trabajo aportamos por primera vez resultados experimentales de la adsorción de ADN lineal sobre monocapas de lípidos zwitteriónicos y aniónicos sin la adición de cationes. Esto se demuestra experimentalmente por medio de monocapas de Langmuir de DOPE/DOPG (1:1) esparcidas sobre una subfase acuosa que contiene ADN de timo de ternera. La adsorción de ADN sobre las monocapas de lípidos zwitteriónicos y aniónicos se discute en función de distintos tipos de electrolitos añadidos. Las medidas del potencial superficial aportan una prueba adicional de la interacción entre el ADN y las monocapas de lípidos zwitteriónicos y aniónicos dependiendo de la presencia y naturaleza del electrolito. A partir de estos resultados determinamos el momento dipolar total perpendicular a las monocapas, permitiendo caracterizar los diferentes tipos de lipoplejos y el papel particular de los lípidos zwitteriónicos.; Anionic lipids are increasingly being used in lipoplexes for synthetic gene vectors as an alternative to cationic lipids. This is primarily due to their lower toxicity, which makes them biocompatible and adaptable to be tissue specific. However, anionic lipoplexes require the presence of multivalent cations to promote the electrostatic attraction between DNA and anionic lipid mono- and bilayers. In this work we provide for the first time experimental results of the adsorption of linear DNA onto anionic/zwitterionic lipid monolayers without any addition of cations. This is demonstrated experimentally by means of Langmuir monolayers of DOPE/DOPG (1:1) lipids spread on a water subphase that contains calf thymus DNA. The adsorption of DNA onto anionic/zwitterionic lipid monolayers is discussed in terms of the surface pressure-molecular area isotherms recorded in the absence and in the presence of different electrolytes. Measurements of the surface potential provide additional evidence of the different interaction of DNA anionic/zwitterionic lipid monolayers depending on the presence and nature of electrolyte. These experimental results are further analysed in terms of the overall dipole moment normal to the monolayers providing new insight into the behaviour of anionic lipoplexes and the role of zwitterionic lipids. https://hdl.handle.net/10481/95576 Discontinuity-enhanced icephobic surfaces for low ice adhesion Ibáñez Ibáñez, Pablo Francisco; Stendardo, Luca; Ospina, Catalina; Chaudhary, Rajat; Tagliaro, Irene; Antonini, Carlo Hypothesis: Passive low ice-adhesion surfaces are frequently composed of soft materials; however, soft materials potentially present durability issues, which could be overcome by fabricating composite surfaces with patterned rigid and soft areas. Here we propose the innovative concept of discontinuity-enhanced icephobic surfaces, where the stress concentration at the edge between rigid and soft areas, i.e. where discontinuities in elasticity are located, facilitates ice detachment. Experiments: Composite model surfaces were fabricated with controlled rigid-soft ratios and discontinuity line lengths. The ice adhesion values were measured while recording the ice/substrate interface, to unravel the underpinning ice detachment mechanism. The experiments were complemented by numerical simulations that provided a better understanding of the ice detachment mechanism. Findings: It was found that when a surface contains rigid and soft areas, stress is concentrated at the edge between soft and hard areas, i.e. at the discontinuity line, rather than all over the soft or rigid areas. An unexpected nonunidirectional crack propagation was observed for the first time and elucidated. When rigid and deformable materials are present, the crack occurs on the discontinuity line and propagates first on rigid and then on soft areas. Moreover, it was demonstrated that an increase in discontinuities promotes crack initiation and leads to a reduction of ice adhesion.