Click Chemistry for Drug Delivery Nanosystems

This is a post-peer-review, pre-copyedit version of an article published in Pharmaceutical Research. The final authenticated version is available online at: https://doi.org/10.1007/s11095-011-0568-5The purpose of this Expert Review is to discuss the impact of click chemistry in nanosized drug delivery systems. Since the introduction of the click concept by Sharpless and coworkers in 2001, numerous examples of click reactions have been reported for the preparation and functionalization of polymeric micelles and nanoparticles, liposomes and polymersomes, capsules, microspheres, metal and silica nanoparticles, carbon nanotubes and fullerenes, or bionanoparticles. Among these click processes, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has attracted most attention based on its high orthogonality, reliability, and experimental simplicity for non-specialists. A renewed interest in the use of efficient classical transformations has been also observed (e.g., thiol-ene coupling, Michael addition, Diels-Alder). Special emphasis is also devoted to critically discuss the click concept, as well as practical aspects of application of CuAAC to ensure efficient and harmless bioconjugationThis work was financially supported by the Spanish Ministry of Science and Innovation (CTQ2009-10963 and CTQ2009-14146-C02-02) and the Xunta de Galicia (10CSA209021PR)S

Messenger RNA delivery by hydrazone-activated polymers

This is the Accepted Manuscript of the following article: Juanes, M., Creese, O., Fernández-Trillo, P., & Montenegro, J. (2019). Messenger RNA delivery by hydrazone-activated polymers. Medchemcomm, 10(7), 1138-1144. doi: 10.1039/c9md00231fThe intracellular delivery of DNA and RNA therapeutics requires the assistance of vectors and/or nucleotide modifications to protect the nucleic acids against host nucleases and promote cellular internalization and release. Recently, messenger RNA (mRNA) has attracted much attention due to its transient activity and lack of genome permanent recombination and persistent expression. Therefore, there is a strong interest in the development of conceptually new non-viral vectors with low toxicity that could improve mRNA transfection efficiency. We have recently introduced the potential of polyhydrazones and the importance of the degree of polymerization for the delivery of siRNA and plasmid DNA. Here, we demonstrate that this technology can be easily adapted to the more interesting complexation and delivery inside living cells of mRNA. The polyplexes resulting from the combination of the amphiphilic polyhydrazone were characterized and the transfection efficiency and cell viability were studied for a discrete collection of functionalized polyhydrazones. The results obtained demonstrated the versatility of these polymeric vectors as excellent candidates for the delivery of mRNA and validate the easy adaptability of the technology to more sensitive and therapeutically relevant nucleic acidsThis work has received financial support from Spanish Agencia Estatal de Investigación (AEI) [CTQ2014-59646-R, SAF2017-89890-R], the Xunta de Galicia (ED431C 2017/25, 2016-AD031 and Centro singular de investigación de Galicia accreditation 2016–2019, ED431G/09) and the European Union (European Regional Development Fund - ERDF). M.J. received a F.P.I. fellowship from MINECO (CTQ2014-59646- R). J.M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP- 677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017). F.F.T. thanks the Birmingham Science City and the European Regional Development Fund, and the University of Birmingham (John Evans Fellowship). O.C. thanks the Midlands Integrative Biosciences Training Partnership (MIBTP) for the PhD scholarshipS

Trust in Leadership and Perceptions of Justice in Fostering Employee Commitment

In a period of uncertainty, trust in leadership and perceptions of fairness have emerged as pivotal factors for fostering employee identification and affective commitment. Drawing from authentic leadership theory, this leadership style is identified as a crucial antecedent of affective commitment, examining the mediating role of distributive justice and the moderating role of interactional justice. A quantitative approach was employed, utilizing data from 302 questionnaires completed by Spanish retail workers. For data analysis, SPSS v.25 was used to generate descriptive statistics, while partial least squares structural equation modeling was applied to test the proposed hypotheses. Our findings revealed that authentic leadership is positively associated with the development of affective commitment, with distributive justice acting as a mediating factor between the two. Furthermore, interactional justice negatively moderates the relationship between distributive justice and affective commitment. Contrary to initial expectations, the second moderation, between authentic leadership and affective commitment, was not found to be significant

Poly(acryloyl hydrazide), a versatile scaffold for the preparation of functional polymers: synthesis and post-polymerisation modification

Here we present the synthesis and post-polymerisation modification of poly(acryloyl hydrazide), a versatile scaffold for the preparation of functional polymers: poly(acryloyl hydrazide) was prepared from commercially available starting materials in a three step synthesis on a large scale, in good yields and high purity. Our synthetic approach included the synthesis of a Boc-protected acryloyl hydrazide, the preparation of polymers via RAFT polymerisation and the deprotection of the corresponding Boc-protected poly(acryloyl hydrazide). Post-polymerisation modification of poly(acryloyl hydrazide) was then demonstrated using a range of conditions for both hydrophilic and hydrophobic aldehydes. These experiments demonstrate the potential of poly(acryloyl hydrazide) as a scaffold in the synthesis of functional polymers, in particular those applications where in situ screening of the activity of the functionalised polymers may be required (e.g. biological applications)This work was supported by the Royal Society, U.K (IE130688) and the Wellcome Trust (177ISSFPP). F. F.-T. thanks the Birmingham Science City and the European Regional Development Fund, the Royal Society (RG140273), and the University of Birmingham (John Evans Fellowship). J. M. thanks funding from MINECO (CTQ2014-59646-R, RYC-2013-1378) the Xunta de Galicia (ED431G/09 and 2016-AD031) and the ERC (Stg-DYNAP-677786)S

Upbringing and parenting. Detection of socio-educational and training needs in parents

Parents often ask themselves what to do and how to deal with the different situations that arise with their children. Parent education programmes (PEP) attempt to provide answers to these questions. Nevertheless, it is pertinent to perform an updated assessment of parents’ training and socio-educational needs in relation to the upbringing and parenting of children. To this end, the following objectives were set: a) to determine the degree of compliance with the principles of positive parenting (PPP) in a sam­ple of mothers and fathers; b) to detect clusters of parents according to PPP and socio-demo­graphic characteristics; c) to identify their pref­erences regarding training and attendance at these interventions. The sample consisted of 389 parents. Qualitative content and quantitative de­scriptive and multivariate cluster analyses were performed and Mann-Whitney U, Kruskal-Wallis H, Binomial and Chi-square tests were applied. Results showed lower scores for communication, stress management and involvement, and high­er scores for shared activities, recognition and affection. Four clusters of parents were obtained (low, medium, high and very high PPP follow-up). They expressed a preference for the group for­mat of the programme, attendance at weekends, in the afternoon and when the children are in early childhood. Some of the training content mentioned was related to emotions, communica­tion, conflict resolution, behaviour management, information and communication technologies, values, norms, sexuality and drugs. Respondents require interventions that take into account the priorities expressed. The findings will allow us to confirm whether the interventions implemented and evaluated in the context explored are a suit­able response to the needs detected, and also to justify the design of any new ones, if necessary.Los progenitores se preguntan en multitud de ocasiones qué y cómo proceder ante las diferentes situaciones surgidas con sus hijos. A estas cuestiones intentan dar respuesta los programas de educación parental (PEP). Sin embargo, es pertinente llevar a cabo un diagnóstico actualizado de necesidades formativas y socioeducativas de los progenitores en relación con la crianza y educación de los menores. Para ello, se establecieron los siguientes objetivos: a) determinar el grado de cumplimiento de los principios de la parentalidad positiva (PPP) en una muestra de padres y madres; b) detectar agrupamientos de progenitores en función de los PPP y las características sociodemográficas; c) identificar sus preferencias formativas y de asistencia a dichas intervenciones. 389 progenitores conformaron la muestra. Se ejecutaron análisis cualitativos de contenidos y cuantitativos descriptivos, así como multivariante de conglomerados; se aplicaron las pruebas U de Mann-Whitney, H de Kruskal-Wallis, Binomial y Chi-cuadrado. Los resultados mostraron menores puntuaciones en comunicación, control del estrés e implicación; mayores en actividades compartidas, reconocimiento y afecto. Se obtuvieron cuatro clústeres de progenitores (bajo, medio, alto y muy alto seguimiento de los PPP). Expusieron predilección por la modalidad grupal de programa, la asistencia los fines de semana, en horario de tarde y cuando los hijos atraviesan la primera infancia. Algunos de los contenidos formativos señalados estaban relacionados con las emociones, la comunicación, la resolución de conflictos, el manejo de comportamientos, las tecnologías de la información y la comunicación, los valores, las normas, la sexualidad y las drogas. Los sujetos encuestados requieren de intervenciones que tengan en cuenta las prioridades expresadas. Los hallazgos permitirán contrastar si las intervenciones implementadas y evaluadas en el contexto explorado contestan con solvencia a las necesidades detectadas, así como justificar el diseño de otras nuevas si fuese necesario

Different-Length Hydrazone Activated Polymers for Plasmid DNA Condensation and Cellular Transfection

The recent advances in genetic engineering demand the development of conceptually new methods to prepare and identify efficient vectors for the intracellular delivery of different nucleotide payloads ranging from short single-stranded oligonucleotides to larger plasmid double-stranded circular DNAs. Although many challenges still have to be overcome, polymers hold great potential for intracellular nucleotide delivery and gene therapy. We here develop and apply the postpolymerization modification of polyhydrazide scaffolds, with different degree of polymerization, for the preparation of amphiphilic polymeric vehicles for the intracellular delivery of a circular plasmid DNA. The hydrazone formation reactions with a mixture of cationic and hydrophobic aldehydes proceed in physiologically compatible aqueous conditions, and the resulting amphiphilic polyhydrazones are directly combined with the biological cargo without any purification step. This methodology allowed the preparation of stable polyplexes with a suitable size and zeta potential to achieve an efficient encapsulation and intracellular delivery of the DNA cargo. Simple formulations that performed with efficiencies and cell viabilities comparable to the current gold standard were identified. Furthermore, the internalization mechanism was studied via internalization experiments in the presence of endocytic inhibitors and fluorescence microscopy. The results reported here confirmed that the polyhydrazone functionalization is a suitable strategy for the screening and identification of customized polymeric vehicles for the delivery of different nucleotide cargosThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [CTQ2014-59646-R, CTQ2016-78423-R, SAF2017-89890-R], the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031), the ERDF, Royal Society U.K (IE130688 and RG140273), and the Wellcome Trust (177ISSFPP). J.M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP-677786), and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017). F. F.-T. thanks the Birmingham Science City and the European Regional Development Fund, and the University of Birmingham (John Evans Fellowship). J.M.P. thanks the Spanish Agencia Estatal de Investigación (AEI) for his research contractS

Nanoparticle transport in epithelial cells: pathway switching through bioconjugation

The understanding and control of nanoparticle transport into and through cellular compartments is central to biomedical applications of nanotechnology. Here, it is shown that the transport pathway of 50 nm polystyrene nanoparticles decorated with vitamin B12 in epithelial cells is different compared to both soluble B12 ligand and unmodified nanoparticles, and this is not attributable to B12 recognition alone. Importantly, the study indicates that vitamin B12-conjugated nanoparticles circumnavigate the lysosomal compartment, the destination of soluble vitamin B12 ligand. Whereas cellular trafficking of soluble B12 is confirmed to occur via the clathrin-mediated pathway, transport of B12-conjugated nanoparticles appears to predominantly take place by a route that is perturbed by caveolae-specific inhibitors. This data suggests that, following its conjugation to nanoparticles, in addition to dramatically increasing the cellular uptake of nanoparticles, the normal cell trafficking of B12 is switched to an alternative pathway, omitting the lysosomal stage: a result with important implications for oral delivery of nanoparticulate diagnostics and therapeutics

Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains

Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4–S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4–S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules

Nanoparticle transport in epithelial cells: pathway switching through bioconjugation

The understanding and control of nanoparticle transport into and through cellular compartments is central to biomedical applications of nanotechnology. Here, it is shown that the transport pathway of 50 nm polystyrene nanoparticles decorated with vitamin B12 in epithelial cells is different compared to both soluble B12 ligand and unmodified nanoparticles, and this is not attributable to B12 recognition alone. Importantly, the study indicates that vitamin B12-conjugated nanoparticles circumnavigate the lysosomal compartment, the destination of soluble vitamin B12 ligand. Whereas cellular trafficking of soluble B12 is confirmed to occur via the clathrin-mediated pathway, transport of B12-conjugated nanoparticles appears to predominantly take place by a route that is perturbed by caveolae-specific inhibitors. This data suggests that, following its conjugation to nanoparticles, in addition to dramatically increasing the cellular uptake of nanoparticles, the normal cell trafficking of B12 is switched to an alternative pathway, omitting the lysosomal stage: a result with important implications for oral delivery of nanoparticulate diagnostics and therapeutics

Rationale and methods of the cardiometabolic valencian study (escarval-risk) for validation of risk scales in mediterranean patients with hypertension, diabetes or dyslipidemia

BackgroundThe Escarval-Risk study aims to validate cardiovascular risk scales in patients with hypertension, diabetes or dyslipidemia living in the Valencia Community, a European Mediterranean region, based on data from an electronic health recording system comparing predicted events with observed during 5 years follow-up study.Methods/DesignA cohort prospective 5 years follow-up study has been designed including 25000 patients with hypertension, diabetes and/or dyslipidemia attended in usual clinical practice. All information is registered in a unique electronic health recording system (ABUCASIS) that is the usual way to register clinical practice in the Valencian Health System (primary and secondary care). The system covers about 95% of population (near 5 million people). The system is linked with database of mortality register, hospital withdrawals, prescriptions and assurance databases in which each individual have a unique identification number. Diagnoses in clinical practice are always registered based on IDC-9. Occurrence of CV disease was the main outcomes of interest. Risk survival analysis methods will be applied to estimate the cumulative incidence of developing CV events over time.DiscussionThe Escarval-Risk study will provide information to validate different cardiovascular risk scales in patients with hypertension, diabetes or dyslipidemia from a low risk Mediterranean Region, the Valencia Community