One and two-photons excitation of aminonaphthalimide–bodipy dyads: biological application

Boron dipyrromethene (BODIPY) dyes are commonly commercialized chromophores that have attracted immense interest as versatile tools in biological chemistry. However, the generally very small Stokes shifts of BODIPY dyes may cause re-absorption or effects from excitation-light scattering, which is a potential drawback for their application, especially in bioimaging. One strategy to avoid this problem is the integration of BODIPY dyes as acceptors in bi- or multichromophoric arrays, some of them qualifying as energy transfer cassettes where donor and acceptor units are electronically non-conjugated. In this communication we present a series of aminonaphthalimide–BODIPY energy transfer cassettes that shown very fast and efficient BODIPY fluorescence sensitization. This was observed upon one- and two-photon excitation, which extends the application range of the investigated bichromophoric dyads in terms of accessible excitation wavelengths. In comparison with the direct excitation of the BODIPY chromophore, the two-photon absorption cross-section of the dyads is significantly incremented by the presence of the aminonaphthalimide.The applicability of the new probes in the one- and two-photon excitation mode was demonstrated, both in solution and supported on silica nanoparticles, in a proof-of-principle approach in the fluorescence imaging of HeLa cells.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Design and synthesis of new dendrons for biomedical applications

Dendrons are excellent multifunctional platforms for many different applications. In particular, the development of multivalent molecular nanostructures, with a well-defined size and shape, has an enormous interest in the field of biomedicine1. Great efforts are being made in the design of new dendritic structures since their precise structure, multivalency and the possibility of functionalizing their terminal groups makes them a useful tool for certain applications. Recently, a new type of dendrimer (dendron) was designed and synthesized based on the iterative coupling of 2,2-Bis (AminoAlkyl) Propanamide units (BAPAD)2,3. This represents a versatile model when incorporating certain functionalities in its structure. However, the synthesis of these new dendritics structures was addressed using 3,3'-dichloropivalic acid as the starting substrate, which translated in the superficial amino groups of these new macromolecule being in relative positions 1,3. For certain applications, where the functionalization of the dendritics structures on its surface requires the introduction of relatively bulky groups, this may result in a problem of reactivity given the steric congestion. Here, we present the design and synthesis of new dendrons where the surface amino groups are in relative positions 1,7. Formally, it involves the preparation of dendrons with longer arms that can minimize the problems of steric congestion. These dendrons can be designed to modify surfaces superficially thanks to the versatility of the carboxylic acid which can be modified to introtoduce in the focal poin diferent functional groups (azido, amido, thiol, etc…).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Synthesis of fluorescent dendrimeric antigen efficiently internalized by human dendritic cells

A new fluorescent dendrimeric antigen (DeAn) based on a dendron with amoxicilloyl terminal groups has been synthetized. The synthesis implies a novel class of all-aliphatic polyamide dendrimer (BisAminoalkylPolyAmide Dendrimers, or BAPAD).[1] The introduction of a cystamine core allows the incorporation of this dendrons into a 1,8-naphthalimide fluorofore functionalized with a maleimide group. The fluorescence properties of this DeAn has been studied and compared with the properties of an equivalent dendron possessing amino-terminal groups. This DeAn has been used as a synthetic antigen in a biomedical assay that tests the amoxicillin sensitivity of dendritic cells (DC) from tolerant and allergic patients.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Managing with (fluorescent)-biomimetic dendrimers

Dendrimers that are modified through their peripheral groups have been employed for many biomedical purposes. Diagnosis via specific interactions with target proteins is one of the objectives that has been given more attention. In this way, we have been developing different methodologies for the detection of immunoglobulins, as an in vitro method for the diagnosis of allergic reactions to drugs [1], [2]. Specific cell interactions of surfaces grafted dendrimers have been applied to the study of cell adhesion. We have studied also the application of dendrimer-based uneven nanopatterns to evaluate the local RGD surface density effects on cell adhesion, as bioactive substrates to evaluate the impact of the RGD local surface density on the chondrogenic induction of adult human mesenchymal stem cells [3], [4], [5]. Although the use of PAMAM dendrimer has allowed us to develop useful methods for this purpose, for certain objectives we have required the development of new dendrimeric models. We have developed a new approach for the production of allaliphatic polyamide dendrimers (BAPAD) by iterative 3,3′-diaminopivalic acid connections as building blocks for dendrimer construction. These dendrimers were studied in explicit solvent by atomistic forcefield-based molecular dynamics to characterize structural properties such as shape, radius and monomer distribution [6]. Fluorescent labeling of these biomimetic dendrimers has been used as a tool to study their interaction with cells.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Preparation, photophysical properties and cell image of Naphthalimide-based fluorescent nanostructures for biomedical applications: BAPAD dendrimeric antigens and silica nanoparticles

We present here the synthesis and characterization of BAPAD [1] fluorescent Dendrimeric-Antigens (DeAn), to be applied in basophil activation as a test to detect drug allergy reactions. A second generation BAPAD dendrimer using cystamine as core that can be reducced to obtain two dendrons with a thiol group in the focal point, useful to attach a fluorescent probe, have been synthesized. The free amine groups on the surface of the dendrimer were functionalized with an amoxiciloyl group (AXO), the allergenic determinant to the beta-lactam antibiotic amoxicillin. The fluorescent DeAn (F-DeAn) has been fully characterized by NMR and MS techniques, and their fluorescent properties well established in aqueous biological media. F-DeAn without the haptenic moieties at the periphery has been also obtained and fully characterized as a control assay. Both molecules have been also characterized using molecular dynamics simulation calculations. We show here how these dendrimeric structures interacts with dendritic cells and are internalized by them. In a secon approximation, Naphthalimide-fluorscent silica nanoparticles have been prepared and characterized, to be used also in basophil activation test.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Diseño y síntesis de nuevos dendrímeros

Los dendrímeros son excelentes plataformas multifuncionales para una gran cantidad de diferentes aplicaciones. En particular, el desarrollo de nanoestructuras moleculares multivalentes, con tamaño y forma bien definida, tiene un enorme interés en el campo de la biomedicina.1 Actualmente resulta necesario poder obtener dendrímeros multifuncionales, con estructuras muy precisas, diseñados específicamente para determinadas aplicaciones. Con las herramientas adecuadas se pueden incluir en la estructura del dendrímero los grupos requeridos en posiciones específicas del mismo. Recientemente se diseñó y sintetizó un nuevo tipo de dendrímero (dendrón) basado en el acoplamiento iterativo de unidades de 2,2-Bis(aminoalquil)propanamidas (BAPAD).2,3 Este representa un modelo versátil a la hora de incorporar determinadas funcionalidades en su estructura. Sin embargo, la síntesis de este nuevo dendrímero se abordó utilizando como sustrato de partida el ácido 3,3´-dicloropiválico. Los grupos amino superficiales de esta nueva macromolécula se encuentran en posiciones relativas 1,3, lo que para determinadas aplicaciones, donde la funcionalización del dendrímero en su superficie requiere introducir grupos relativamente voluminosos, supone un problema de reactividad dado la congestión estérica que se provoca. Se presenta el diseño y la síntesis de nuevos dendrímeros donde los grupos amino superficiales, necesarios para determinadas aplicaciones, queden en posiciones relativas 1,7. Formalmente implica la preparación de dendrímeros con brazos más largos que podrán minimizar los problemas de congestión estérica.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Diseño y síntesis de nuevos dendrímeros y dendrones. Aplicaciones en regeneración tisular

Los dendrímeros han generado un enorme interés debido a su particular estructura ramificada y su multivalencia. La extensa investigación que se ha llevado a cabo sobre este tipo de macromoléculas ha demostrado la naturaleza multidisciplinar de las mismas, describiéndose sus aplicaciones en química, física, biología, medicina o en la mejora de nuevos materiales. En particular, el desarrollo de nanoestructuras moleculares multivalentes, con tamaño y forma bien definida, tiene un enorme interés en el campo de la biomedicina. El empleo de estos sistemas como plataformas multifuncionales en el estudio de las reacciones adversas a fármacos RAF (alergia a antibióticos betalactámicos en particular) ha quedado ampliamente demostrado. La síntesis de una serie de Antígenos Dendriméricos (DeAn) y su anclaje sobre distintas superficies, ha permitido avanzar en el estudio del diagnóstico clínico de la alergia a antibióticos betalactámicos mediante la técnica de Radio Allergo Sorbent Test (RAST). Recientemente, se han iniciado trabajos enfocados hacia la aplicación de estas macromoléculas en otra área de la biomedicina. En particular, se han realizado estudios sobre los procesos de adhesión celular enfocados al empleo de células madres mesenquimales (MSC) en regeneración tisular. La incorporación de patrones de reconocimiento celular basados en el péptido RGD (Arginina-Glicina-Ácido aspártico) a estructuras dendriméricas provoca un aumento de la adhesión celular in vitro, observando no sólo un aumento de la adhesión de las células a la superficie sino que la eficacia de esta adhesión está relacionada con la forma en que el dendrímero presenta el tripéptido a la célula. El empleo de plataformas sólidas que contengan las estructuras dendriméricas funcionalizadas puede ser crucial para determinadas aplicaciones en regeneración tisular.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Slightly congested amino terminal dendrimers. The synthesis of amide-based stable structures on a large scale

Nowadays, amino terminal dendrimers are appealing materials for biological applications due to their multivalence and the versatile conjugation of the amino groups. However, the high reactivity of these terminal groups can be decreased by steric hindrance, limiting their possible bioapplications. Herein, we report the divergent synthesis of slightly sterically hindered amino terminal polyamide dendrimers. A simple and unique AB2 scaffold has been chosen to build the dendritic structures, where only amide bonds have been used as the connecting unit. The 1–7 relative positions of the amino groups in the AB2 monomers avoid the steric congestion of the macromolecules, allowing the construction of robust dendrimers up to the fifth generation. The construction of the dendrimers is based on two well-established reactions, using simple and cheap reactants, with yields above 90% on a gram scale and easy purification procedures. This synthetic methodology constitutes an easy and efficient way for the preparation of stable and aqueous soluble dendrimers on a gram scale, representing a substantial improvement over the synthesis of this kind of aliphatic polyamide amino terminal dendrimer. The prepared structures were completely characterized and evaluated by size exclusion chromatography, diffusion ordered spectroscopy and atomic force microscopy to determine their size. Molecular dynamics simulations were also carried out and the values obtained were consistent with the experimentally determined values.This work was supported by the Ministerio de Ciencia y Educación (PID2019-104293GB-I00), Ministerio de Ciencia e Innovación (Proyectos de I+D+I “Programación Conjunta Internacional”), EuroNanoMed 2019 (PCI2019-111825-2), Instituto de Salud Carlos III (ISCIII; RETIC ARADYAL RD16/0006/0012) and Junta de Andalucía and Universidad de Málaga (UMA18-FEDERJA-007). We gratefully acknowledge the computer resources provided by the SCBI (Supercomputing and Bioinformatics Center) of the University of Malaga. NMR experiments have been performed in the ICTS “NANBIOSIS”, in the U28 Unit at the Andalusian Centre for Nanomedicine and Biotechnology (BIONAND)

Diseño de dendrímeros amino-terminales basados en enlaces tipo amidas. Estructuras con menor repulsión estérica y síntesis a gran escala

La estructura ramificada y la elevada multivalencia que presentan los dendrímeros los convierten en excelentes compuestos con potenciales aplicaciones en el campo de la biomedicina. En concreto, los dendrímeros con grupos amino terminales han resultado ser una herramienta muy útil en este campo gracias a la gran versatilidad que presentan. Nuestro grupo de investigación tiene una gran experiencia en el diseño y síntesis de dendrímeros amino-terminales [1]. La inserción de estas unidades dendriméricas en complejos luminiscentes nos ha permitido obtener macromoléculas con excelentes propiedades como biomarcadores [2]. Además, la modificación superficial de implantes orales de titanio con este tipo de estructuras ha contribuido a mejorar la biocompatibilidad de los mismos [3]. En esta comunicación presentamos una nueva familia de dendrímeros de poliamida amino-terminales. Su diseño implica una mayor separación entre los grupos terminales, generando así estructuras con menor congestión estérica. La metodología sintética se basa en procedimientos bien establecidos, utilizando reactivos sencillos y económicos, con rendimientos superiores al 90%, en escala de gramos y evitando procedimientos de purificación tediosos [4