Cyclodextrin-Based Nanostructure Efficiently Delivers siRNA to Glioblastoma Cells Preferentially via Macropinocytosis

© 2020 by the authors.Small interfering ribonucleic acid (siRNA) has the potential to revolutionize therapeutics since it can knockdown very efficiently the target protein. It is starting to be widely used to interfere with cell infection by HIV. However, naked siRNAs are unable to get into the cell, requiring the use of carriers to protect them from degradation and transporting them across the cell membrane. There is no information about which is the most efficient endocytosis route for high siRNA transfection efficiency. One of the most promising carriers to efficiently deliver siRNA are cyclodextrin derivatives. We have used nanocomplexes composed of siRNA and a β-cyclodextrin derivative, AMC6, with a very high transfection efficiency to selectively knockdown clathrin heavy chain, caveolin 1, and p21 Activated Kinase 1 to specifically block clathrin-mediated, caveolin-mediated and macropinocytosis endocytic pathways. The main objective was to identify whether there is a preferential endocytic pathway associated with high siRNA transfection efficiency. We have found that macropinocytosis is the preferential entry pathway for the nanoparticle and its associated siRNA cargo. However, blockade of macropinocytosis does not affect AMC6-mediated transfection efficiency, suggesting that macropinocytosis blockade can be functionally compensated by an increase in clathrin- and caveolin-mediated endocytosis.This research was funded, in part, by contract numbers PID2019-105858RB-I00 (MCI, AEI, FEDER, UE) and RTI2018-097609-B-C21 (MICIU, AEI, FEDER, UE) to C.O.M. and J.M.G.F. and grants from the Spanish Ministerio de Economía y Competitividad (project SAF2017-89288-R from MINECO/AEI/FEDER/UE), from Junta de Comunidades de Castilla-La Mancha (project SBPLY/19/180501/000067), and ISCIII (AC19/00075) and ERANET Euronanomed Program (project NANO4GLIO) to V.C. It also benefited from the framework of COST Action Nano2Clinic (CA17140), supported by COST (European Cooperation in Science and Technology).Publisher’s versio

Trehalose-based Janus cyclooligosaccharides: the ‘‘Click’’ synthesis and DNA-directed assembly into pH-sensitive transfectious nanoparticles

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.-- et al.The convergent preparation of Janus molecular nanoparticles by thiourea-‘‘clicking’’ of a,a0 -trehalose halves has been implemented; the strategy allows access to macrocyclic derivatives with seggregated cationic and lipophilic domains that in the presence of DNA undergo pH-dependent self-assembly into lamellar superstructures, as established by electrochemical, structural (SAXS), microscopical (TEM) and computational techniques, that mediate transfection in vitro and in vivo.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).The authors thank MINECO (contract numbers CTQ2012- 30821, SAF2013-44021-R, CTQ2015-64425-C2-1-R and CTQ2015-64425-C2-2-R), the Junta de Andalucía (contract number FQM2012-1467), University Complutense of Madrid (project no. UCMA05-33-010), the Government of Navarra (Department of Innovation and Industry, contract number IIQ14334.RI1), the University of Navarra Foundation (FUN), and the European Regional Development Funds (FEDER and FSE) for financial support.Peer reviewe

Evaluation of the automatic ELISA Triturus analyser

The objective was to evaluate the automatic ELISA Triturus analyser in order to assess its practicability and imprecision. The analyser was evaluated according to the guidelines of the Spanish Society of Clinical Chemistry. The evaluation was performed in two steps: evaluation of the imprecision and the inaccuracy of a semiquantitative and qualitative technique, and study of the practicability. The within-run imprecision rate ranged from 8.9 (VC) to 10.2% (VC) for the semiquantitative test, and from 11 (VC) to 17.2% (VC) for the qualitative one. The between-run imprecision rate ranged from 6 (VC) to 9.7% (VC) for the semiquantitative test, and from 8.8 (VC) to 18%(VC) for the qualitative technique. No sample-related carryover was detected in negative samples. The relative inaccuracy was correct for both techniques. Non-specific binding caused by waste products from the analysed circuits was not detected. The Triturus analyser is a reliable open analytical system with a considerable working capability, rendering this apparatus adequate for conventional laboratories with a medium-to-high work charge

Comparative studies on lectin–carbohydrate interactions in low and high density homo- and heteroglycoclusters

12 páginas, 5 figuras, 3 tablas, 3 esquemas.A versatile synthetic procedure to construct series of high- and low-density homo- and heteroglycoclusters is reported. The binding properties of these synthetic multivalent glycoconjugates to concanavalin A (Con A), a model lectin, have been assessed by using a range of competitive and non-competitive binding assays including enzyme-linked lectin assays (ELLA), isothermal titration microcalorimetry (ITC) and surface plasmon resonance (SPR). In all cases, highly dense glycoclusters showed a substantial amplification of the lectin-binding strength in comparison with low-density counterparts. Interestingly, highly-dense glycoligand presentations, regardless of their homo- or heteroglycoligand pattern, furnished similar Con A binding properties, supporting the existence of a synergic effect (heterocluster effect) due to secondary interactions of “non-active” structural motifs in the presence of a certain density of “active” glycoligands.We thank the Spanish Ministerio de Educación y Ciencia (contracts number CTQ2006-15515-C02-01/BQUand CTQ2007-61180/PPQ) and the Junta de Andalucía for financial support.Peer reviewe

Enhanced Gene Delivery Triggered by Dual pH/Redox Responsive Host-Guest Dimerization of Cyclooligosaccharide Star Polycations

A robust strategy is reported to build perfectly monodisperse star polycationscombining a trehalose-based cyclooligosaccharide (cyclotrehalan, CT) centralcore onto which oligoethyleneimine radial arms are installed. Thearchitectural perfection of the compounds is demonstrated by a variety ofphysicochemical techniques, including NMR, MS, DLS, TEM, and GPC. Key tothe strategy is the possibility of customizing the cavity size of the macrocyclicplatform to enable/prevent the inclusion of adamantane motifs. Theseproperties can be taken into advantage to implement sequential levels ofstimuli responsiveness by combining computational design, precisionchemistry and programmed host-guest interactions. Specifically, it is shownthat supramolecular dimers implying a trimeric CT-tetraethyleneimine starpolycation and purposely designed bis-adamantane guests are preorganizedto efficiently complex plasmid DNA (pDNA) into transfection-competentnanocomplexes. The stability of the dimer species is responsive to theprotonation state of the cationic clusters, resulting in dissociation at acidicpH. This process facilitates endosomal escape, but reassembling can takeplace in the cytosol then handicapping pDNA nuclear import. By equippingthe ditopic guest with a redox-sensitive disulfide group, recapturingphenomena are prevented, resulting in drastically improved transfectionefficiencies both in vivo and in vitro.Ministerio de Ciencia e Innovación y Agencia Estatal de Investigación, de España-MCIN/AEI/10.13039/501100011033Fondo Europeo de Desarrollo Regional de la Unión Europea-RTI2018-097609-B-C21, RTI2018-097609-B-C22, PID2019-105858RB-I00 y PID2020-118384GB-I00Junta de Andalucía-P20_0016

Trifaceted Mickey Mouse Amphiphiles for Programmable Self-Assembly, DNA Complexation and Organ-Selective Gene Delivery

Instilling segregated cationic and lipophilic domains with an angular disposition in a trehalose-based trifaceted macrocyclic scaffold allows engineering patchy molecular nanoparticles leveraging directional interactions that emulate those controlling self-assembling processes in viral capsids. The resulting trilobular amphiphilic derivatives, featuring a Mickey Mouse architecture, can electrostatically interact with plasmid DNA (pDNA) and further engage in hydrophobic contacts to promote condensation into transfectious nanocomplexes. Notably, the topology and internal structure of the cyclooligosaccharide/pDNA co-assemblies can be molded by fine-tuning the valency and characteristics of the cationic and lipophilic patches, which strongly impacts the transfection efficacy in vitro and in vivo. Outstanding organ selectivities can then be programmed with no need of incorporating a biorecognizable motif in the formulation. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes by making cyclooligosaccharide patchiness the focus.Ministerio de Ciencia, Innovación y Universidades y Agencia Estatal de Investigación de España. RTI2018-097609-B-C21, RTI2018-097609-B-C22 y PID2019-105858RB-I00Universidad de Alcalá de Henares, Madrid. CCG19/CC-03

Identifcation of Loci Associated with Susceptibility to Bovine Paratuberculosis and with the Dysregulation of the MECOM, eEF1A2, and U1 Spliceosomal RNA Expression

Although genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with the susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection, only a few functional mutations for bovine paratuberculosis (PTB) have been characterized. Expression quantitative trait loci (eQTLs) are genetic variants typically located in gene regulatory regions that alter gene expression in an allele-specific manner. eQTLs can be considered as functional links between genomic variants, gene expression, and ultimately phenotype. In the current study, peripheral blood (PB) and ileocecal valve (ICV) gene expression was quantified by RNA-Seq from fourteen Holstein cattle with no lesions and with PTB-associated histopathological lesions in gut tissues. Genotypes were generated from the Illumina LD EuroG10K BeadChip. The associations between gene expression levels (normalized read counts) and genetic variants were analyzed by a linear regression analysis using R Matrix eQTL 2.2. This approach allowed the identification of 192 and 48 cis-eQTLs associated with the expression of 145 and 43 genes in the PB and ICV samples, respectively. To investigate potential relationships between these cis-eQTLs and MAP infection, a case-control study was performed using the genotypes for all the identified cis-eQTLs and phenotypical data (histopathology, ELISA for MAP-antibodies detection, tissue PCR, and bacteriological culture) of 986 culled cows. Our results suggested that the heterozygous genotype in the cis-eQTL-rs43744169 (T/C) was associated with the up-regulation of the MDS1 and EVI1 complex (MECOM) expression, with positive ELISA, PCR, and bacteriological culture results, and with increased risk of progression to clinical PTB. As supporting evidence, the presence of the minor allele was associated with higher MECOM levels in plasma samples from infected cows and with increased MAP survival in an ex-vivo macrophage killing assay. Moreover, the presence of the two minor alleles in the cis-eQTL-rs110345285 (C/C) was associated with the dysregulation of the eukaryotic elongation factor 1-alpha2 (eEF1A2) expression and with increased ELISA (OD) values. Finally, the presence of the minor allele in the cis-eQTL rs109859270 (C/T) was associated with the up-regulation of the U1 spliceosomal RNA expression and with an increased risk of progression to clinical PTB. The introduction of these novel functional variants into marker-assisted breeding programs is expected to have a relevant effect on PTB control.Financial support for this study was provided by a grant from the Spanish Ministry of Science, Innovation, and Universities (MICINN, https://sede.micinn.gob.es/, project code: RTI2018-094192-R-C21) and by European Regional Development Funds (FEDER) to MAH. This study was co-funded by a grant from the Plan of Science, Technology, and Innovation of the Principality of Asturias, Regional funds PCTI 2018–2020 (www.ficyt.es/pcti/), project code: IDI2018-000237. MC and CBV have been awarded fellowships from the National Institute for Agricultural Research (INIA) progra

Trifaceted Mickey Mouse Amphiphiles for Programmable Self-Assembly, DNA Complexation and Organ-Selective Gene Delivery

Instilling segregated cationic and lipophilic domains with an angular disposition in a trehalose-based trifaceted macrocyclic scaffold allows engineering patchy molecular nanoparticles leveraging directional interactions that emulate those controlling self-assembling processes in viral capsids. The resulting trilobular amphiphilic derivatives, featuring a Mickey Mouse architecture, can electrostatically interact with plasmid DNA (pDNA) and further engage in hydrophobic contacts to promote condensation into transfectious nanocomplexes. Notably, the topology and internal structure of the cyclooligosaccharide/pDNA co-assemblies can be molded by fine-tuning the valency and characteristics of the cationic and lipophilic patches, which strongly impacts the transfection efficacy in vitro and in vivo. Outstanding organ selectivities can then be programmed with no need of incorporating a biorecognizable motif in the formulation. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes by making cyclooligosaccharide patchiness the focus.Peer reviewe

International conference on the healthy effect of virgin olive oil

Ageing represents a great concern in developed countries because the number of people involved and the pathologies related with it, like atherosclerosis, morbus Parkinson, Alzheime's disease, vascular dementia, cognitive decline, diabetes and cancer. Epidemiological studies suggest that a Mediterranean diet (which is rich in virgin olive oil) decreases the risk of cardiovascular disease. The Mediterranean diet, rich in virgin olive oil, improves the major risk factors for cardiovascular disease, such as the lipoprotein profile, blood pressure, glucose metabolism and antithrombotic profile. Endothelial function, inflammation and oxidative stress are also positively modulated. Some of these effects are attributed to minor components of virgin olive oil. Therefore, the definition of the Mediterranean diet should include virgin olive oil. Different observational studies conducted in humans have shown that the intake of monounsaturated fat may be protective against age-related cognitive decline and Alzheimer's disease. Microconstituents from virgin olive oil are bioavailable in humans and have shown antioxidant properties and capacity to improve endothelial function. Furthermore they are also able to modify the haemostasis, showing antithrombotic properties. In countries where the populations fulfilled a typical Mediterranean diet, such as Spain, Greece and Italy, where virgin olive oil is the principal source of fat, cancer incidence rates are lower than in northern European countries. The protective effect of virgin olive oil can be most important in the first decades of life, which suggests that the dietetic benefit of virgin olive oil intake should be initiated before puberty, and maintained through life. The more recent studies consistently support that the Mediterranean diet, based in virgin olive oil, is compatible with a healthier ageing and increased longevity. However, despite the significant advances of the recent years, the final proof about the specific mechanisms and contributing role of the different components of virgin olive oil to its beneficial effects requires further investigations. © 2005 Blackwell Publishing Ltd