PAMAM-based dendrimers for diverse biomedical applications

Existe una gran necesidad de nuevos tratamientos en determinadas patologías tales como el cáncer o las infecciones resistentes. Entre algunas de las nuevas terapias más prometedoras se encuentran génica y celular, pero ambas tienen serios problemas que resolver antes de pasar a la clínica. Para resolver algunos de estos problemas, recientemente se han combinado vectores virales con células madre (células madre mesenquimales especialmente). Sin embargo, ya que son células complejas de crecer con los métodos y medios de cultivo estándar, la técnica tiene que ser mejorado antes de ser poder ser utilizado en ensayos clínicos. Vectores sintéticos tales como polímeros, aunque más seguros que los virus, generalmente no poseen la eficacia requerida para los tratamiento de terapia génica. En este ámbito, los dendrímeros son polímeros sintéticos tridimensionales, altamente ramificados y con una estructura química bien definida. En particular, los dendrímeros basados en PAMAM (poli(amidoamina)) han sido ampliamente investigados por sus aplicaciones biológicas y han mostrado ser portadores de propiedades clínicamente relevantes que les hacen valiosos para ser utilizados en terapia génica o celular. Se ha demostrado que los dendrímeros basados en PAMAM también pueden facilitar la localización de antibióticos en el interior de bacterias, y por lo tanto, pueden ser utilizados para mejorar la actividad antimicrobiana. Además, pueden ser altamente tóxicos para determinadas bacterias mediante la disrupción de la membrana citoplasmática. Controlando la funcionalidad y carga a través de la elección de los grupos periféricos, el tamaño del dendrímero y el nivel de recubrimiento con PEG, se pueden modular las interacciones con la superficie bacteriana y su actividad antibacteriana y citotóxica para las células huésped. En este proyecto se han estudiado las propiedades biomédicas de derivados de PAMAM y su influencia sobre el crecimiento bacteriano y celular. Nuestros resultados muestran que, aunque el dendrímero PAMAM completamente recubierto con PEG disminuye el crecimiento de Pseudomonas y que tiene potencial para ser empleado como un agente antibacteriano, en el caso de las células madre mesenquimales, el crecimiento celular se incrementa sin alterar sus patrones de pluripotencialidad y podría ser de gran ayuda para las futuras estrategias de terapia celular

SARS-CoV-2 mutant spectra reveal differences between COVID-19 severity categories

Trabajo presentado en el XVI Congreso Nacional de Virología, celebrado en Málaga (España) del 06 al 09 de septiembre de 2022.RNA virus populations are composed of complex mixtures of genomes that are termed mutant spectra. SARS-CoV-2 replicates as a viral quasispecies, and mutations that are detected at low frequencies in a host can be dominant in subsequent variants. We have studied mutant spectrum complexities of SARS-CoV-2 populations derived from thirty nasopharyngeal swabs of patients infected during the first wave (April 2020) in the Hospital Universitario Fundación Jiménez Díaz. The patients were classified according to the COVID-19 severity in mild (non-hospitalized), moderate (hospitalized) and exitus (hospitalized with ICU admission and who passed away due to COVID-19). Using ultra-deep sequencing technologies (MiSeq, Illumina), we have examined four amplicons of the nsp12 (polymerase)-coding region and two amplicons of the spike-coding region. Ultra-deep sequencing data were analyzed with different cut-off frequency for mutation detection. Average number of different point mutations, mutations per haplotype and several diversity indices were significantly higher in SARS-CoV-2 isolated from patients who developed mild disease. A feature that we noted in the SARS-CoV-2 mutant spectra from diagnostic samples is the remarkable absence of mutations at intermediate frequencies, and an overwhelming abundance of mutations at frequencies lower than 10%. Thus, the decrease of the cut-off frequency for mutation detection from 0.5% to 0.1% revealed an increasement (50- to 100 fold) in the number of different mutations. The significantly higher frequency of mutations in virus from patients displaying mild than moderate or severe disease was maintained with the 0.1% cut- off frequency. To evaluate whether the frequency repertoire of amino acid substitutions differed between SARS-CoV-2 and the well characterized hepatitis C virus (HCV), we performed a comparative study of mutant spectra from infected patients using the same bioinformatics pipelines. HCV did not show the deficit of intermediate frequency substitutions that was observed with SARS-CoV-2. This difference was maintained when two functionally equivalent proteins, the corresponding viral polymerases, were compared. In conclusion, SARS-CoV-2 mutant spectra are rich reservoirs of mutants, whose complexity is not uniform among clinical isolates. Virus from patients who developed mild disease may be a source of new variants that may acquire epidemiological relevance.This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and In-novation (COVID-19 Research Call COV20/00181), and co-financed by European Development Regional Fund ‘A way to achieve Europe’. The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P., M.C., and P.M. are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006, and CP16/00116, respectively) co-financed by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investi-gación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE). R.L.-V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. C.G.-C. is sup-ported by predoctoral contract PRE2018-083422 from MCIU. BS was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181), and co-financed by European Development Regional Fund ‘A way to achieve Europe’. The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P., M.C., and P.M. are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006, and CP16/00116, respectively) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE). R.L.-V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. P.S. is supported by postdoctoral contract “Margarita Salas” CA1/RSUE/2021 from MCIU. B.S. was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO.Peer reviewe

Derivados de BoltornTM decorados con dendrones de bis-MPA para aplicaciones en terapia génica

Resumen del póster presentado a la 6ª Jornada de Jóvenes Investigadores en Física y Química de Aragón celebrada en Zaragoza el 20 de noviembre de 2014.Este trabajo ha sido financiado por el proyecto del MINECO CTQ2012-35692.Peer reviewe

Litchi

Treball Final de Grau en Comunicació Audiovisual. Modalitat C – Curtmetratge surrealista. Codi: CA0932. Curs acadèmic 2020-2021Litchi es un cortometraje surrealista realizado por los cuatro miembros del equipo que conforman este Trabajo Final de Grado: Arnau Pastor, Irene Sifre, Alba Regina González y Rebeca Hornes. Este cuenta la historia de Ismael y Carlota, una pareja joven que llevan unos años juntos, sumergidos en una relación amorosa basada en la monotonía y la dependencia, y por consiguiente, tóxica. Se ha escogido realizar un cortometraje surrealista porque este movimiento siempre ha atraído a los integrantes de Litchi. Además, el formato con el que se trabaja (el vídeo) permite ampliamente reflejar mucha creatividad con esta historia, siendo así el formato audiovisual que más les atrae. Litchi es el resultado del trabajo en equipo, de una buena organización y de la unión de diferentes perfiles en un mismo grupo. De esta manera, sus integrantes han dado lo mejor de ellos mismos, ofreciendo y demostrando sus mejores facetas técnicas y artísticas. Con esta obra se ha querido demostrar los objetivos desarrollados posteriormente en este trabajo, que se engloban en dos puntos. Por un lado, trabajar en equipo de forma eficiente y comunicativa para conseguir un resultado óptimo y por otro lado, mostrar la historia de la manera más creativa, poniendo en práctica aquello que se ha aprendido a lo largo de la carrera. Litchi ofrece la posibilidad de enfrentarse a situaciones peliagudas, complicadas y desafiantes; puesto que ellos acaban de salir de la carrera de Comunicación Audiovisual y este es el primer proyecto profesional al que se enfrentan. Así, el equipo de Litchi ha querido plasmar los conocimientos adquiridos durante la carrera de Comunicación Audiovisual, siendo conscientes de que este proyecto es un antes y después en su vida profesional. El resultado fílmico final ha sido mejor de lo esperado, demostrando así que con esfuerzo y dedicación todo se puede cumplir con creces. Este proyecto ha conseguido aumentar la autoestima de todos los componentes del equipo técnico, demostrando a cada uno que son capaces de lograr un relato audiovisual grande, óptimo y exitoso.Litchi is a surrealist short-film made by the four members of the team that work on this end-of-degree project: Arnau Pastor, Irene Sifre, Alba Regina González and Rebeca Hornes. This short-film tells the story of Ismael and Carlota, a young couple who have been together for a few years, immersed in a love relationship that is based on monotony and dependency, and therefore: toxicity. They have chosen to work on a surrealistic short-film because this cultural movement has always attracted the members of Litchi. In addition, the format that they are working on (video), allows us to reflect a lot of creativity in this story. Also, the audiovisual format is the one that attracts us the most. Litchi is the result of teamwork, a good organization and the union of different profiles in the same group. In this way, its members have given the best of themselves, offering and demonstrating their best technical and artistic aspects. Throughout this project they have tried to accomplish the objectives that they have set. They are developed later through these pages, which are included in two points. On the one hand, working as a team in an efficient and communicative way to achieve an optimal result. And on the other hand, relate the story in the most creative way, putting into practice what has been learned throughout the career. Litchi could be a very challenging project, as it can come up with tricky and complicated situations; since they have just graduated from the Audiovisual Communication degree and this is the first professional project they face. Thus, the Litchi team wanted to capture the knowledge acquired during the Audiovisual Communication degree, being aware that this project is a before and after in their professional life. The final film result has been better than expected, thus demonstrating that with effort and dedication everything can be more than achieved. This project has managed to increase the self-esteem of all the components of the technical team, showing each one that they are capable of achieving a great, optimal and successful audiovisual story

Derivados dendríticos con núcleo de BoltornTM como agentes de transfección

Resumen del trabajo presentado al V Encuentro sobre Dendrímeros, celebtrado en Zaragoza (España) del 25 al 26 de enero de 2016.La terapia génica constituye un área de gran interés, debido a su potencial para prevenir o tratar numerosas enfermedades mediante la introducción de genes terapéuticos en las células y tejidos. Aunque los vectores virales muestran una alta eficacia, su producción es cara y pueden producir reacciones inmunes adversas y toxicidad en el hígado. Los sistemas de transfección no virales ofrecen una alternativa no inmunogénica, pero su eficacia es baja. En el caso de los sistemas más utilizados, como los polímeros catiónicos (polientilenimina o PEI), o los dendrímeros catiónicos (PAMAM y PPI), mantener el balance entre una eficacia elevada vs citotoxicidad es complicado. Los sistemas dendríticos basados en 2,2-bis(methylol)propionic acid (bis-MPA) son una buena alternativa ya que son polyesters biodegradables, biocompatibles tanto in vitro como in vivo, solubles en un entorno biológico y fáciles de funcionalizar. Dentro de este grupo, los BoltornTM son polímeros hiperramificados más sencillos de sintetizar que los dendrímeros. En este trabajo se presentan una serie de derivados con núcleo de BoltornTM recubiertos con dendrones de bis-MPA funcionarizados con glicinas, con el objetivo de aumentar el número de grupos amino terminales que contribuyen con sus cargas positivas al aumento de la capacidad de formar complejos con el DNA6 y a una mejor internalización y transfección.Este trabajo ha sido financiado por MINECO (proyecto CTQ2012-35692), DGA-FSE (grupo E04), UFV 2013-PMD.Peer reviewe

Nanoobjects formed by ionic PAMAM dendrimers: hydrophilic/lipophilic modulation and encapsulation properties

In this paper, we present the preparation and properties of some ionic PAMAM derivatives, which combine hydrophilic and lipophilic carboxylic acid chains as counter-ions for all protonable inner and outer amino groups. The amphiphilic nature of the final ionic codendrimers and, hence, their self-assembling features can be modulated by using different ratios between hydrophilic and lipophilic chains. In the bulk, these new materials self-organize into smectic A liquid crystal phases. In water, they self-assemble into different types of nano-objects depending on the molecular composition. The study of the morphology of these nano-structures, their cytotoxicity and their capability to encapsulate a lipophilic anticancer drug are reported herein. Some of these nanoobjects are non-cytotoxic and present good drug trapping ability, which make them interesting nanocarriers for applications in nanotechnology and biomedicine.This work was supported by the FP7 PEOPLE PROGRAMME, the Marie Curie Actions; ITN, no. 215884-2, the MINECO, Spain (under Projects: CTQ2012-35692 and MAT2012-38538-CO3-01), which included FEDER funding, and the Aragón Government FSE (Project E04). Thanks are given to: Nuclear Magnetic Resonance, Mass Spectrometry and Thermal Analysis Services from the CEQMA, Universidad de Zaragoza-CSIC (Spain). A.L. thanks the MECD (Spain) for FPU grants. E.F. is thankful for the support from the EU (Marie Curie ITN ‘Dendreamers’) as an ESR. R.G.-P. is thankful for the support from the Aragón PhD student program (DGA).Peer Reviewe

Complexing the Oncolytic Adenoviruses Ad∆∆ and Ad-3∆-A20T with Cationic Nanoparticles Enhances Viral Infection and Spread in Prostate and Pancreatic Cancer Models

Oncolytic adenoviruses (OAd) can be employed to efficiently eliminate cancer cells through multiple mechanisms of action including cell lysis and immune activation. Our OAds, AdΔΔ and Ad-3∆-A20T, selectively infect, replicate in, and kill adenocarcinoma cells with the added benefit of re-sensitising drug-resistant cells in preclinical models. Further modifications are required to enable systemic delivery in patients due to the rapid hepatic elimination and neutralisation by blood factors and antibodies. Here, we show data that support the use of coating OAds with gold nanoparticles (AuNPs) as a possible new method of virus modification to help augment tumour uptake. The pre-incubation of cationic AuNPs with AdΔΔ, Ad-3∆-A20T and wild type adenovirus (Ad5wt) was performed prior to infection of prostate/pancreatic cancer cell lines (22Rv, PC3, Panc04.03, PT45) and a pancreatic stellate cell line (PS1). Levels of viral infection, replication and cell viability were quantified 24–72 h post-infection in the presence and absence of AuNPs. Viral spread was assessed in organotypic cultures. The presence of AuNPs significantly increased the uptake of Ad∆∆, Ad-3∆-A20T and Ad5wt in all the cell lines tested (ranging from 1.5-fold to 40-fold), compared to virus alone, with the greatest uptake observed in PS1, a usually adenovirus-resistant cell line. Pre-coating the AdΔΔ and Ad-3∆-A20T with AuNPs also increased viral replication, leading to enhanced cell killing, with maximal effect in the most virus-insensitive cells (from 1.4-fold to 5-fold). To conclude, the electrostatic association of virus with cationic agents provides a new avenue to increase the dose in tumour lesions and potentially protect the virus from detrimental blood factor binding. Such an approach warrants further investigation for clinical translation

Gold nanoparticle coatings as efficient adenovirus carriers to non-infectable stem cells

Mesenchymal stem cells (MSCs) are adult pluripotent cells with the plasticity to be converted into different cell types. Their self-renewal capacity, relative ease of isolation, expansion and inherent migration to tumors, make them perfect candidates for cell therapy against cancer. However, MSCs are notoriously refractory to adenoviral infection, mainly because CAR (Coxsackie-Adenovirus Receptor) expression is absent or downregulated. Over the last years, nanoparticles have attracted a great deal of attention as potential vehicle candidates for gene delivery, but with limited effects on their own. Our data showed that the use of positively charged 14 nm gold nanoparticles either functionalized with arginine–glycine–aspartate (RGD) motif or not, increases the efficiency of adenovirus infection in comparison to commercial reagents without altering cell viability or cell phenotype. This system represents a simple, efficient and safe method for the transduction of MSCs, being attractive for cancer gene and cell therapies.This work was supported by Araid Fund; by the Government of Aragon through a PhD grant (B054/12); Fundación Mutua Madrileña Automovilística (MMA); Instituto de Salud Carlos III, and ERANET-NANOSCIERA NANOTRUCK projects.Peer reviewe

Gold nanoparticle coatings as efficient adenovirus carriers to non-infectable stem cells

Mesenchymal stem cells (MSCs) are adult pluripotent cells with the plasticity to be converted into different cell types. Their self-renewal capacity, relative ease of isolation, expansion and inherent migration to tumors, make them perfect candidates for cell therapy against cancer. However, MSCs are notoriously refractory to adenoviral infection, mainly because CAR (Coxsackie-Adenovirus Receptor) expression is absent or downregulated. Over the last years, nanoparticles have attracted a great deal of attention as potential vehicle candidates for gene delivery, but with limited effects on their own. Our data showed that the use of positively charged 14 nm gold nanoparticles either functionalized with arginine–glycine–aspartate (RGD) motif or not, increases the efficiency of adenovirus infection in comparison to commercial reagents without altering cell viability or cell phenotype. This system represents a simple, efficient and safe method for the transduction of MSCs, being attractive for cancer gene and cell therapies.This work was supported by Araid Fund; by the Government of Aragon through a PhD grant (B054/12); Fundación Mutua Madrileña Automovilística (MMA); Instituto de Salud Carlos III, and ERANET-NANOSCIERA NANOTRUCK projects.Peer reviewe