Properties of polyplexes formed between a cationic polymer derived from l-arabinitol and nucleic acids

In this work a sugar-based cationic polymer derived from l-arabinitol, PUArab, was prepared and its interactions with the linear calf thymus DNA and with the circular plasmid pEGFP-C1 were investigated at different N/P ratios. The polyplexes were characterized by using several techniques. For both nucleic acids, a charge inversion was observed, together with a conformational change from a coiled structure to a more compacted one. However, the N/P ratio required to observe the DNA condensation depended on the nucleic acid architecture. PUArab presents low toxicity in several cell lines. The transfection efficiency, TE, of the PUArab/pEGFP-C1 polyplexes was investigated at several N/P ratios in order to study their potential as vectors in gene transfection

Multivalent Calixarene-Based Liposomes as Platforms for Gene and Drug Delivery

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4 , were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drugThis work was financed by the Consejería de Conocimiento, Innovación y Universidades de la Junta de Andalucía (FQM-206, FQM-274, and PY20-01234), the VI Plan Propio Universidad de Sevilla (PP2019/00000748), RTI2018-100692-B-100; P18-RT-1271; PI18-0005-2018; VI-PP AY.SUPLEM2019; RYC-2015-18670, The R+D+I grant PID2019-104195G from the Spanish Ministry of Science and Innovation-Agencia Estatal de Investigación/10.13039/501100011033 (P.H.) and the European Union (Feder Funds). The authors thank the University of Seville for the grant VPPI-US. J.A.L. also thanks the Fundación ONCE funded by the Fondo Social Europe

Self-aggregation in aqueous solution of amphiphilic cationic calix[4]arenes. Potential use as vectors and nanocarriers

The self-aggregation of four amphiphilic cationic calix[4]arenes, CALIX, in aqueous solutions was investigated in this work. The nature of the polar group present at the upper rim as well as the length of the hydrophobic tails attached to the lower rim was varied. All the calixarenes present two critical aggregation concentrations, CAC1 and CAC2. For [CALIX] CAC2 micelles and a wide distribution of vesicles were found. Cell viability experiments show that calixarene micelles and several of the calixarene vesicles investigated could be used as biocompatible nanocarriers. On this basis, the study of the interactions between the cationic calixarene aggregates (micelles and vesicles) and calf thymus DNA, ctDNA, were done and the results indicated that most of them strongly interact with the polynucleotide, inverting its charge. Micelles totally compact the ctDNA, while vesicles only partially cause conformational changes in the nucleic acid. Therefore, the CALIX micelles show potential as vectors in gene therapy. The encapsulation of the antineoplastic drug doxorubicin into the calixarene aggregates was also investigated. A high encapsulation efficiency was found for micelles and, specially, for vesicles. However, DOX-loaded calixarene vesicles present low stability at 37 °C, which is a serious restriction in their use as nanocarriers for this drug. The release of DOX from the calixarene micelles shows that they could lengthen the half-life of free doxorubicin in the body and, as a result, lower amounts of drug could be used in the cancer treatments diminishing the important side effects of DOX.This work was supported by the Consejería de Educación y Ciencia de la Junta de Andalucía (P12-FQM-1105, FQM-206 and FQM-274, and PI-0005-2018), the VI Plan Propio Universidad de Sevilla (PP2018-10338), the Ministerio de Ciencia, Innovación y Universidades (RTI2018-100692-B-I00), the grant Ramon y Cajal RYC2015-1867 and the European Union (Feder Funds). The authors thank the University of Seville for the grant VPPI-US

FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine

Fanconi anemia (FA) is a rare hereditary disorder caused by mutations in any one of the FANC genes. FA cells are mainly characterized by extreme hypersensitivity to interstrand crosslink (ICL) agents. Additionally, the FA proteins play a crucial role in concert with homologous recombination (HR) factors to protect stalled replication forks. Here, we report that the 5-methyl-2’-deoxycytidine (5mdC) demethylation (pathway) intermediate 5-hydroxymethyl-2’-deoxycytidine (5hmdC) and its deamination product 5-hydroxymethyl-2’-deoxyuridine (5hmdU) elicit a DNA damage response, chromosome aberrations, replication fork impairment and cell viability loss in the absence of FANCD2. Interestingly, replication fork instability by 5hmdC or 5hmdU was associated to the presence of Poly(ADP-ribose) polymerase 1 (PARP1) on chromatin, being both phenotypes exacerbated by olaparib treatment. Remarkably, Parp1 cells did not show any replication fork defects or sensitivity to 5hmdC or 5hmdU, suggesting that retained PARP1 at base excision repair (BER) intermediates accounts for the observed replication fork defects upon 5hmdC or 5hmdU incorporation in the absence of FANCD2. We therefore conclude that 5hmdC is deaminated in vivo to 5hmdU, whose fixation by PARP1 during BER, hinders replication fork progression and contributes to genomic instability in FA cells.The eHAP cell line were a generous gift from Dr. G. Crossan (MRC-LMB, Cambridge). DT40, NV012 and HSC72 cell lines were kindly provided by K.J. Patel (MRC WIMM, Oxford), and the MDA-MB-231, MDA-MB-436 and DLD1 BRCA2−/− were a gift from Dr. Pablo Huertas and Dr. Andrés Aguilera (CABIMER, Sevilla) respectively. We are in debt to Dr. J.A. Pérez-Simón (IBIS, Seville) for providing hosting and helpful advice. IVR´s lab is funded by MCIN/AEI/ 10.13039/501100011033, grant 18.06.03.3073 (RTI2018-100692-B-100), Consejerías de Salud, y de Economía y Conocimiento de la Junta de Andalucía, grants number PI-0005-2018, and 18.06.03.2404 (P18-RT-1271), Universidad de Sevilla “Programa Operativo FEDER Andalucía 2014–2020”, grant number 18.06.03.2319 (US-1381081), and by “ERDF A way of making Europe”. IVR is a recipient of Ramón y Cajal Contracts RYC2015-18670. SK lab was supported by ERC-2016-AdG/742654 grant

Preparation and characterization of metallomicelles of Ru(II). Cytotoxic activity and use as vector

The use of nanovectors in several medicinal treatments has reached a great importance in the last decade. Some drugs need to be protected to increase their lifetimes in the blood flow, to avoid degradation, to be delivered into target cells or to decrease their side effects. The goal of this work was to design and prepare nanovectors formed by novel surfactants derived from the [Ru(bpy)3]2+ complex. These amphiphilic molecules are assembled to form metallomicelles which can act as pharmaceutical agents and, at the same time, as nanovectors for several drugs. TEM images showed a structural transition from spherical to elongated micelles when the surfactant concentration increased. Fluorescence microscopy confirmed the internalization of these metallomicelles into diverse cell lines and cytotoxicity assays demonstrated specificity for some human cancer cells. The encapsulation of various antibiotics was carried out as well as a thorough study about the DNA condensation by the metallomicelles. To the best of our knowledge, applications of these metallomicelles have not been shown in the literature yet.This work was supported by the Consejería de Educación y Ciencia de la Junta de Andalucía (Proyecto de Excelencia P12-FQM-1105, FQM-206 and FQM-274), the VI Plan Propio Universidad de Sevilla (2018/500) and the European Union (Feder Funds). The authors thank the University of Seville for the grant VPPI-US.Peer reviewe

Genotoxicity of tetrahydrofolic acid to hematopoietic stem and progenitor cells.

Metabolically reactive formaldehyde is a genotoxin and a carcinogen. Mice lacking the main formaldehyde-detoxifying gene Adh5 combined with the loss of the Fanconi anemia (FA) DNA repair pathway rapidly succumbed to bone marrow failure (BMF) primarily due to the extensive ablation of the hematopoietic stem cell (HSC) pool. However, the mechanism by which formaldehyde mediates these toxic effects is still unknown. We uncover a detrimental role of tetrahydrofolic acid (THF) in cells lacking Adh5 or the FA repair pathway. We show that Adh5- or FA-deficient cells are hypersensitive to formaldehyde and to THF, presenting DNA damage and genome instability. THF cytotoxicity involved imbalance of the nucleotide pool by deregulation of the thymidylate synthase (TYMS) enzyme, which stalled replication forks. In mice, THF exposure had widespread effects on hematopoiesis, affecting the frequency and the viability of myeloid- and lymphoid-committed precursor cells. Moreover, the hematopoietic stem and progenitor cells (HSPC) showed genomic instability, reduced colony-forming capacity and increased frequency of cycling and apoptotic HSCs upon THF exposure. Overall, our data reveal that the physiological pool of THF and formaldehyde challenge the stability of the genome of HSPCs that might lead to blood disorders