Electroactive nanoinjection platform for intracellular delivery and gene silencing

Background: Nanoinjection—the process of intracellular delivery using vertically configured nanostructures—is a physical route that efficiently negotiates the plasma membrane, with minimal perturbation and toxicity to the cells. Nanoinjection, as a physical membrane-disruption-mediated approach, overcomes challenges associated with conventional carrier-mediated approaches such as safety issues (with viral carriers), genotoxicity, limited packaging capacity, low levels of endosomal escape, and poor versatility for cell and cargo types. Yet, despite the implementation of nanoinjection tools and their assisted analogues in diverse cellular manipulations, there are still substantial challenges in harnessing these platforms to gain access into cell interiors with much greater precision without damaging the cell’s intricate structure. Here, we propose a non-viral, low-voltage, and reusable electroactive nanoinjection (ENI) platform based on vertically configured conductive nanotubes (NTs) that allows for rapid influx of targeted biomolecular cargos into the intracellular environment, and for successful gene silencing. The localization of electric fields at the tight interface between conductive NTs and the cell membrane drastically lowers the voltage required for cargo delivery into the cells, from kilovolts (for bulk electroporation) to only ≤ 10 V; this enhances the fine control over membrane disruption and mitigates the problem of high cell mortality experienced by conventional electroporation. Results: Through both theoretical simulations and experiments, we demonstrate the capability of the ENI platform to locally perforate GPE-86 mouse fibroblast cells and efficiently inject a diverse range of membrane-impermeable biomolecules with efficacy of 62.5% (antibody), 55.5% (mRNA), and 51.8% (plasmid DNA), with minimal impact on cells’ viability post nanoscale-EP (> 90%). We also show gene silencing through the delivery of siRNA that targets TRIOBP, yielding gene knockdown efficiency of 41.3%. Conclusions: We anticipate that our non-viral and low-voltage ENI platform is set to offer a new safe path to intracellular delivery with broader selection of cargo and cell types, and will open opportunities for advanced ex vivo cell engineering and gene silencing. Graphical abstract: [Figure not available: see fulltext.

3 years of liraglutide versus placebo for type 2 diabetes risk reduction and weight management in individuals with prediabetes: a randomised, double-blind trial

Background: Liraglutide 3·0 mg was shown to reduce bodyweight and improve glucose metabolism after the 56-week period of this trial, one of four trials in the SCALE programme. In the 3-year assessment of the SCALE Obesity and Prediabetes trial we aimed to evaluate the proportion of individuals with prediabetes who were diagnosed with type 2 diabetes. Methods: In this randomised, double-blind, placebo-controlled trial, adults with prediabetes and a body-mass index of at least 30 kg/m2, or at least 27 kg/m2 with comorbidities, were randomised 2:1, using a telephone or web-based system, to once-daily subcutaneous liraglutide 3·0 mg or matched placebo, as an adjunct to a reduced-calorie diet and increased physical activity. Time to diabetes onset by 160 weeks was the primary outcome, evaluated in all randomised treated individuals with at least one post-baseline assessment. The trial was conducted at 191 clinical research sites in 27 countries and is registered with ClinicalTrials.gov, number NCT01272219. Findings: The study ran between June 1, 2011, and March 2, 2015. We randomly assigned 2254 patients to receive liraglutide (n=1505) or placebo (n=749). 1128 (50%) participants completed the study up to week 160, after withdrawal of 714 (47%) participants in the liraglutide group and 412 (55%) participants in the placebo group. By week 160, 26 (2%) of 1472 individuals in the liraglutide group versus 46 (6%) of 738 in the placebo group were diagnosed with diabetes while on treatment. The mean time from randomisation to diagnosis was 99 (SD 47) weeks for the 26 individuals in the liraglutide group versus 87 (47) weeks for the 46 individuals in the placebo group. Taking the different diagnosis frequencies between the treatment groups into account, the time to onset of diabetes over 160 weeks among all randomised individuals was 2·7 times longer with liraglutide than with placebo (95% CI 1·9 to 3·9, p<0·0001), corresponding with a hazard ratio of 0·21 (95% CI 0·13–0·34). Liraglutide induced greater weight loss than placebo at week 160 (–6·1 [SD 7·3] vs −1·9% [6·3]; estimated treatment difference −4·3%, 95% CI −4·9 to −3·7, p<0·0001). Serious adverse events were reported by 227 (15%) of 1501 randomised treated individuals in the liraglutide group versus 96 (13%) of 747 individuals in the placebo group. Interpretation: In this trial, we provide results for 3 years of treatment, with the limitation that withdrawn individuals were not followed up after discontinuation. Liraglutide 3·0 mg might provide health benefits in terms of reduced risk of diabetes in individuals with obesity and prediabetes. Funding: Novo Nordisk, Denmark

Improving fracture properties of MEMS components by surface control

This thesis studies the mechanical reliability of nanostructures. The strength statistics of Si nanobeams, their dependence on surface morphology and degradation due to air exposure are characterized and necessary conditions for maximum strength and durability are determined. Due to their small sizes and use of low defect materials, nanostructures have the potential to be used in applications requiring very high stresses at low failure probabilities. Fracture strength of 190-nm thick Si beams have been shown to be as high as 13 GPa, approximately 30 times higher than the strength of macroscale samples. Testing similarly prepared beams etched with relatively smooth morphologies (0.4 nm rms) we showed that the strengths were further improved to 16 GPa, approaching theoretical strengths predicted by previous atomistic calculations. To explain this influence, a series of fracture mechanics based Monte Carlo simulations were performed. Chemically modified surfaces of the tested beams were measured, statistically characterized and equivalent surfaces were generated. The surfaces consisted of bunched steps which act as stress concentrators, resulting in very high local stresses and hence enhancing material failure. Simulations of nanobeams processed using two different chemical etchants demonstrate the impact of surface morphology on fracture strengths characterized in terms of the Weibull distribution. It was shown that even a small increase in roughness reduces the strength considerably. This high strength potential is promising for nanomechanical devices requiring high stress levels. Yet, for practical applications, maintenance of strength throughout the structure's service life may be as important as high initial strengths. Tests performed over a period of three weeks showed that this high strength degrades to 11 GPa when the beams are exposed to air. Coating the sample surfaces with protective methyl monolayers resulted in a 10\% higher initial mean strength, which was maintained throughout the test period under the same environmental conditions as the uncoated samples. Our results show that the strength degradation can be prevented by effective protection of surfaces. The results of our experiments and simulations suggest that surface control is essential for the improvement and maintenance of high mechanical strengths at nanoscales.Cornell Center for Materials Research (CCMR), a Materials Research Science and Engineering Center of the National Science Foundation (DMR-0520404

Physics Based Formulation of a Cohesive Zone Model for Ductile Fracture

This paper addresses a physics based derivation of mode-I and mode-II traction separation relations in the context of cohesive zone modeling of ductile fracture of metallic materials. The formulation is based on the growth of an array of pores idealized as cylinders which are considered as therepresentative volume elements. An upper bound solution is applied for the deformation of the representative volume element and different traction-separation relations are obtained through different assumptions

Micromechanical cohesive zone relations for ductile fracture

This paper addresses the derivation of a micromechanically motivated incremental mixed-mode traction separation law in the context of cohesive zone modeling of crack propagation in ductile metallic materials. The formulation is based on the growth of an array of pores idealized as cylinders which are considered as the representative volume elements. An upper bound solution is applied for the deformation of the representative volume element and different incremental traction-separation relations are obtained for mixed-mode loading conditions. While most of the current traction-separation relations used in cohesive zone modeling consider phenomenological relations, in the current work micromechanical parameters such as size, shape and spacing of pores describe the level of damage and linkage of the pores characterizes the propagating crack. Copyright (C) 2016 The Authors. Published by Elsevier B.V

Energy-based non-local plasticity models for deformation patterning, localization and fracture

This paper analyses the effect of the form of the plastic energy potential on the (heterogeneous) distribution of the deformation field in a simple setting where the key physical aspects of the phenomenon could easily be extracted. This phenomenon is addressed through two different (rate-dependent and rate-independent) non-local plasticity models, by numerically solving two distinct one-dimensional problems, where the plastic energy potential has different non-convex contributions leading to patterning of the deformation field in a shear problem, and localization, resulting ultimately in fracture, in a tensile problem. Analytical and numerical solutions provided by the two models are analysed, and they are compared with experimental observations for certain cases