PEG–Polypeptide Block Copolymers as pH-Responsive Endosome-Solubilizing Drug Nanocarriers

Herein we report the potential of click chemistry-modified polypeptide-based block copolymers for the facile fabrication of pH-sensitive nanoscale drug delivery systems. PEG–polypeptide copolymers with pendant amine chains were synthesized by combining N-carboxyanhydride-based ring-opening polymerization with post-functionalization using azide–alkyne cycloaddition. The synthesized block copolymers contain a polypeptide block with amine-functional side groups and were found to self-assemble into stable polymersomes and disassemble in a pH-responsive manner under a range of biologically relevant conditions. The self-assembly of these block copolymers yields nanometer-scale vesicular structures that are able to encapsulate hydrophilic cytotoxic agents like doxorubicin at physiological pH but that fall apart spontaneously at endosomal pH levels after cellular uptake. When drug-encapsulated copolymer assemblies were delivered systemically, significant levels of tumor accumulation were achieved, with efficacy against the triple-negative breast cancer cell line, MDA-MB-468, and suppression of tumor growth in an in vivo mouse model.Novartis Institutes of Biomedical ResearchNational Institutes of Health (U.S.) (Centers for Cancer Nanotechnology Excellence Grant P30 CA14051)National Institutes of Health (U.S.) (Centers for Cancer Nanotechnology Excellence Grant 5 U54 CA151884-02)National Science Foundation (U.S.). Graduate Research FellowshipNatural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship

Layer-by-Layer Assembled Antisense DNA Microsponge Particles for Efficient Delivery of Cancer Therapeutics

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.United States. Dept. of Defense. Ovarian Cancer Research Program (Teal Innovator Award Grant OC120504)Natural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award 1F32EB017614-01)National Science Foundation (U.S.). Graduate Research Fellowshi

Mesoporous Silica Nanoparticles Loaded with Surfactant: Low Temperature Magic Angle Spinning 13C and 29Si NMR Enhanced by Dynamic Nuclear Polarization

We show that dynamic nuclear polarization (DNP) can be used to enhance NMR signals of13C and 29Si nuclei located in mesoporous organic/inorganic hybrid materials, at several hundreds of nanometers from stable radicals (TOTAPOL) trapped in the surrounding frozen disordered water. The approach is demonstrated using mesoporous silica nanoparticles (MSN), functionalized with 3-(N-phenylureido)propyl (PUP) groups, filled with the surfactant cetyltrimethylammonium bromide (CTAB). The DNP-enhanced proton magnetization is transported into the mesopores via 1H–1H spin diffusion and transferred to rare spins by cross-polarization, yielding signal enhancements εon/off of around 8. When the CTAB molecules are extracted, so that the radicals can enter the mesopores, the enhancements increase to εon/off ≈ 30 for both nuclei. A quantitative analysis of the signal enhancements in MSN with and without surfactant is based on a one-dimensional proton spin diffusion model. The effect of solvent deuteration is also investigated

Layer-by-Layer Nanoparticles for Systemic Codelivery of an Anticancer Drug and siRNA for Potential Triple-Negative Breast Cancer Treatment

A single nanoparticle platform has been developed through the modular and controlled layer-by-layer process to codeliver siRNA that knocks down a drug-resistance pathway in tumor cells and a chemotherapy drug to challenge a highly aggressive form of triple-negative breast cancer. Layer-by-layer films were formed on nanoparticles by alternately depositing siRNA and poly-l-arginine; a single bilayer on the nanoparticle surface could effectively load up to 3500 siRNA molecules, and the resulting LbL nanoparticles exhibit an extended serum half-life of 28 h. In animal models, one dose via intravenous administration significantly reduced the target gene expression in the tumors by almost 80%. By generating the siRNA-loaded film atop a doxorubicin-loaded liposome, we identified an effective combination therapy with siRNA targeting multidrug resistance protein 1, which significantly enhanced doxorubicin efficacy by 4 fold in vitro and led to up to an 8-fold decrease in tumor volume compared to the control treatments with no observed toxicity. The results indicate that the use of layer-by-layer films to modify a simple liposomal doxorubicin delivery construct with a synergistic siRNA can lead to significant tumor reduction in the cancers that are otherwise nonresponsive to treatment with Doxil or other common chemotherapy drugs. This approach provides a potential strategy to treat aggressive and resistant cancers, and a modular platform for a broad range of controlled multidrug therapies customizable to the cancer type in a singular nanoparticle delivery system.Janssen Pharmaceutical Ltd. (TRANSCEND Grant)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Health and Medical Research Council (Australia) (CJ Martin Fellowship)National Science Foundation (U.S.). Graduate Research FellowshipNatural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship

Community assessment to advance computational prediction of cancer drug combinations in a pharmacogenomic screen

The effectiveness of most cancer targeted therapies is short-lived. Tumors often develop resistance that might be overcome with drug combinations. However, the number of possible combinations is vast, necessitating data-driven approaches to find optimal patient-specific treatments. Here we report AstraZeneca’s large drug combination dataset, consisting of 11,576 experiments from 910 combinations across 85 molecularly characterized cancer cell lines, and results of a DREAM Challenge to evaluate computational strategies for predicting synergistic drug pairs and biomarkers. 160 teams participated to provide a comprehensive methodological development and benchmarking. Winning methods incorporate prior knowledge of drug-target interactions. Synergy is predicted with an accuracy matching biological replicates for >60% of combinations. However, 20% of drug combinations are poorly predicted by all methods. Genomic rationale for synergy predictions are identified, including ADAM17 inhibitor antagonism when combined with PIK3CB/D inhibition contrasting to synergy when combined with other PI3K-pathway inhibitors in PIK3CA mutant cells.Peer reviewe

O ensino da leitura e da escrita no 1º ano de escolaridade: os resultados dos alunos em leitura

Tese de Doutoramento em Psicologia Aplicada, Área de especialidade Psicologia EducacionalO presente estudo teve como objetivo caracterizar as práticas de ensino da linguagem escrita em Portugal e perceber o seu impacto na aquisição da leitura no final do 1º ano de escolaridade. A primeira etapa deste estudo correspondeu a um estudo descritivo e comparativo com o realizado por Fijalkow (2003) em França. A partir da resposta a um questionário de 883 professores foi possível verificar que os professores dos dois países se aproximam no que diz respeito aos aspetos metodológicos do ensino da leitura e à avaliação da leitura e da escrita dos alunos. Contudo, os professores portugueses dizem utilizar com maior frequência do que os professores franceses, atividades a partir de livros infantis, outros materiais para além do manual e diferentes propostas de escrita. Na segunda etapa deste estudo, o questionário utilizado foi validado através da sua estrutura fatorial e foram utilizados os fatores revelados para a realização de uma análise hierárquica de clusters. Esta análise revelou três grupos de professores: a) um grupo de professores que centra as suas práticas no ensino do código da linguagem escrita (unidades curtas); b) um grupo que realça a construção de significado na interação com o material escrito (unidades longas); c) e um grupo de professores que reúne nas suas práticas características de ambos os grupos referidos anteriormente (unidades diversificadas). A terceira etapa realizada diz respeito à validação das respostas do questionário através da observação de sala de aula. Foram selecionados 5% dos professores de cada um dos três grupos constituídos (N=42). Cada professor foi observado duas vezes durante 60 minutos em situações escolhidas por si como situações privilegiadas para o ensino da linguagem escrita. Através da utilização de uma grelha de observação que divide as atividades desenvolvidas em atividades que remetem para o código ou para o significado, foi possível confirmar as respostas ao questionário para 55% dos professores participantes. No sentido de perceber o impacto das práticas dos professores na aquisição da leitura dos alunos foram selecionados os professores cujas observações foram coincidentes com as respostas ao questionário e avaliados os seus alunos com recurso a três provas de leitura: leitura de palavras isoladas, decisão lexical e compreensão. Participaram 461 alunos distribuídos pelos três grupos referidos anteriormente. Através da realização de uma MANCOVA, controlando a escolaridade das mães dos alunos, verificou-se que os alunos do grupo unidades diversificadas obtêm melhores resultados em todas as provas do que os alunos dos outros dois grupos. Foi ainda possível constatar que não existiram diferenças de desempenho entre os alunos do grupo unidades curtas e os alunos do grupo unidades longas.ABSTRACT: The present study aimed to characterize the teaching practices of written language and understand its impact on reading acquisition by the end of 1st grade. The first step of this study was a descriptive and comparative study to the one conducted by Fijalkow (2003) in France. Based on questionnaire answers of 883 teachers, we found that teachers from both countries have similar approaches regarding methodological aspects of teaching of reading and students’ assessment of reading and writing abilities. However, Portuguese teachers stated that they use more frequently activities from children's literature, other materials besides the manual and different writing proposals than French teachers. In the second step of the study, the questionnaire was validated through its factorial structure and the factors disclosed were used to perform a hierarchical cluster analysis. This analysis revealed three groups of teachers: a) a group of teachers who focus its practices in teaching the code of written language (short units), b) a group that emphasizes the construction of meaning in interaction with the written material (long units), c) and a group of teachers who gathered in their practices characteristics of both groups mentioned above (diversified units). The third step concerns the validation of the questionnaire answers through classroom observation. 5% of the teachers in each of the three groups were selected (N=42). Each teacher was observed twice during 60 minutes in situations identified by them as privileged situations for teaching written language. Using an observation grid which divides the activities conducted in code-activities or the meaning-activities it was possible to confirm questionnaire answers for 55% of the participating teachers. In order to understand the impact of teachers’ practices in students reading acquisition, teachers whose observations were coincident with the questionnaire answers were selected and their students were evaluated using three reading tests: single words reading, lexical decision task and comprehension task. Participants were 461 students distributed through the three groups previously mentioned. By conducting a MANCOVA, controlling mothers’ education level, we concluded that students in the diversified unit group obtain better results in all reading tests than students from the other groups. It was also possible to confirm that there were no differences in performance between students of the short units group and students of the long units group.Tese de Doutoramento em Psicologia Aplicada, Área de especialidade Psicologia EducacionalApoio financeiro da Fundação para a Ciência e Tecnologia (SFRH/BD/64630/2009

Stereochemical plasticity modulates cooperative binding in a CoII12L6 cuboctahedron

Biomolecular receptors are able to process information by responding differentially to combinations of chemical signals. Synthetic receptors that are likewise capable of multi-stimuli response can form the basis of programmable molecular systems, wherein specific input sequences create distinct outputs. Here we report a pseudo-cuboctahedral assembly capable of cooperatively binding anionic and neutral guest species. The binding of pairs of fullerene guests was observed to effect the all-or-nothing cooperative templation of an S6-symmetric host stereoisomer. This bis-fullerene adduct exhibits different cooperativity in binding pairs of anions from the fullerene-free parent: in one case, positive cooperativity is observed, while in another all binding affinities are enhanced by an order of magnitude, and in a third the binding events are only minimally perturbed. This intricate modulation of binding affinity, and thus cooperativity, renders our new cuboctahedral receptor attractive for incorporation into systems with complex, programmable responses to different sets of stimuli.This work was supported by the UK Engineering and Physical Sciences Research Council (EPSRC). F.J.R. acknowledges Cambridge Australia Scholarships and the Cambridge Trust for PhD funding