Quantifying the Average Number of Nucleic Acid Therapeutics per Nanocarrier by Single Particle Tracking Microscopy.

Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies.

Photothermally Triggered Endosomal Escape and Its Influence on Transfection Efficiency of Gold-Functionalized JetPEI/pDNA Nanoparticles.

Plasmonic nanoparticles for drug delivery have attracted increasing interest over the last few years. Their localized surface plasmon resonance causes photothermal effects on laser irradiation, which allows for delivering drugs in a spatio-temporally controlled manner. Here, we explore the use of gold nanoparticles (AuNP) as carriers for pDNA in combination with pulsed laser irradiation to induce endosomal escape, which is currently considered to be one of the major bottlenecks in macromolecular drug delivery on the intracellular level. In particular, we evaluate nanocomplexes composed of JetPEI (polyethylenimine)pDNA and 10 nm AuNP, which do not exhibit endosomal escape by themselves. After incubating HeLa cells with these complexes, we evaluated endosomal escape and transfection efficiency using low- and high-energy laser pulses. At low laser energy heat is produced by the nanocomplexes, while, at higher laser energy, explosive vapour nanobubbles (VNB) are formed. We investigated the ability of heat transfer and VNB formation to induce endosomal escape and we examine the integrity of pDNA cargo after inducing both photothermal effects. We conclude that JetPEI/pDNA/AuNP complexes are unable to induce meaningful transfection efficiencies because laser treatment causes either dysfunctionality of the cargo when VNB are formed or forms too small pores in the endosomal membrane to allow pDNA to escape in case of heating. We conclude that laser-induced VNB is the most suitable to induce effective pDNA endosomal escape, but a different nanocomplex structure will be required to keep the pDNA intact.

Vapor nanobubble is the more reliable photothermal mechanism for inducing endosomal escape of siRNA without disturbing cell homeostasis.

Strategies for controlled delivery of therapeutic siRNA into living cells are in high demand as endosomal escape remains the most prominent bottleneck at the intracellular level. Photothermal properties of gold nanoparticles (AuNP) can be used to overcome the endosomal membrane barrier upon laser irradiation by two mechanisms: endosomal rupture by mechanical energy from water vapor nanobubbles (VNBs), or permeabilization of the endosomal membrane by heat diffusion. Here we evaluated how both mechanisms influence cargo release, transfection efficiency, acute cytotoxicity and cell homeostasis. Using a siRNA/AuNP drug delivery system we found that the in vitro release of siRNA from the AuNP carrier occurs equally efficiently by VNB formation or heat generation. Heat-mediated endosomal escape happened more efficiently in cells that had more particles per endosome, resulting in variable siRNA-induced downregulation (20-50%). VNB-mediated endosomal escape did not dependent on the number of AuNP per endosome, yielding high downregulations (50-60%) independent of the cell type. Effects on cell homeostasis by whole transcriptome analysis, showed a quick recover after 24 h or 48 h for either of both photothermal mechanisms. We conclude that VNBs are more consistent to induce efficient endosomal escape and gene silencing independent of the cell type without long lasting effects on cell homeostasis.

The proton sponge hypothesis: Fable or fact?

In non-viral gene therapy, cationic polymers and lipids are frequently used to encapsulate macromolecular therapeutics into nanoparticles. During their journey to deliver the cargo to the intended intracellular target, many biological barriers need to be overcome. One of the major bottlenecks for efficient transfection is the endosomal barrier since nanoparticles often remain entrapped inside endosomes and are trafficked towards the lysosomes where the cargo is degraded. For cationic polymers, the proton sponge hypothesis was introduced in the late '90s as a way to explain their endosomal escape properties. However, to date, no consensus has been reached in the scientific community about the validity of this hypothesis due to many contradictory reports. Here we review the sometimes conflicting reports that have been published on the proton sponge hypothesis. We also discuss membrane destabilization and polymer swelling as additional factors that might influence endosomal escape of polyplexes. Based on the key publications on this subject, we aim to launch a consensus on the role of the proton sponge hypothesis in endosomal escape.

Endosomal Size and Membrane Leakiness Influence Proton Sponge-Based Rupture of Endosomal Vesicles.

In gene therapy, endosomal escape represents a major bottleneck since nanoparticles often remain entrapped inside endosomes and are trafficked toward the lysosomes for degradation. A detailed understanding of the endosomal barrier would be beneficial for developing rational strategies to improve transfection and endosomal escape. By visualizing individual endosomal escape events in live cells, we obtain insight into mechanistic factors that influence proton sponge-based endosomal escape. In a comparative study, we found that HeLa cells treated with JetPEI/pDNA polyplexes have a 3.5-fold increased endosomal escape frequency compared to ARPE-19 cells. We found that endosomal size has a major impact on the escape capacity. The smaller HeLa endosomes are more easily ruptured by the proton sponge effect than the larger ARPE-19 endosomes, a finding supported by a mathematical model based on the underlying physical principles. Still, it remains intriguing that even in the small HeLa endosomes, <10% of the polyplex-containing endosomes show endosomal escape. Further experiments revealed that the membrane of polyplex-containing endosomes becomes leaky to small compounds, preventing effective buildup of osmotic pressure, which in turn prevents endosomal rupture. Analysis of H1299 and A549 cells revealed that endosomal size determines endosomal escape efficiency when cells have comparable membrane leakiness. However, at high levels of membrane leakiness, buildup of osmotic pressure is no longer possible, regardless of endosomal size. Based on our findings that both endosomal size and membrane leakiness have a high impact on proton sponge-based endosomal rupture, we provide important clues toward further improvement of this escape strategy.

Theory-of-mind, cognitive development, and children's interpretation of anxiety-related physical symptoms.

The present study investigated developmental patterns in children's interpretation of anxiety-related physical symptoms and emotional reasoning, and to what extent these phenomena are influenced by children's level of cognitive development. A large sample of 4-13-year-old children (N=358) were exposed to vignettes in which the presence and absence of physical symptoms was systematically varied. In addition, children completed a series of conservation tasks and a theory-of-mind-test. Results demonstrated that from the age of 7, children were increasingly able to link physical symptoms to anxiety. Furthermore, cognitive development appeared to enhance children's ability to interpret physical symptoms as a sign of anxiety. Further, children's tendency to infer danger from vignettes with physical symptoms (i.e., emotional reasoning) was already prominent in 4-6-year-olds. The implications for physical symptom-based theories of childhood anxiety are briefly discussed.

Effect of video lag on laparoscopic surgery: correlation between performance and usability at low latencies.

BACKGROUND: Few telesurgery studies assess the impact of latency on user experience, low latencies are often not studied despite evidence of negative effects, and some studies recruit inexperienced subjects instead of surgeons without evidence that latency affects both groups similarly. METHODS: Fifteen trainees and fourteen laparoscopic surgeons conducted two tasks on a laparoscopy home-trainer at six latencies below 200 milliseconds (ms). Completion time and usability (perceived awareness of latency, inefficiency, disturbance, adaptability, and impact on patient safety) were measured. RESULTS: Weak correlation between completion time and usability was found. There was significant deterioration in performance and user experience at 105 ms added latency. Surgeons were more negatively affected. CONCLUSION: Objective measures insufficiently describe the impact of latency therefore standard measures of user experience should be incorporated in studies. Even low latencies may be detrimental to laparoscopic surgery. Results from non-experts cannot predict the impact of latency on experienced surgeons. Copyright © 2016 John Wiley & Sons, Ltd.

Design of smart GE11-PLGA/PEG-PLGA blend nanoparticulate platforms for parenteral administration of hydrophilic macromolecular drugs: synthesis, preparation and in vitro/ex vivo characterization.

Active drug targeting and controlled release of hydrophilic macromolecular drugs represent crucial points in designing efficient polymeric drug delivery nanoplatforms. In the present work EGFR-targeted polylactide-co-glycolide (PLGA) nanoparticles were made by a blend of two different PLGA-based polymers. The first, GE11-PLGA, in which PLGA was functionalized with GE11, a small peptide and EGFR allosteric ligand, able to give nanoparticles selective targeting features. The second polymer was a PEGylated PLGA (PEG-PLGA) aimed at improving nanoparticles hydrophilicity and stealth features. GE11 and GE11-PLGA were custom synthetized through a simple and inexpensive method. The nanoprecipitation technique was exploited for the preparation of polymeric nanoparticles composed by a 1:1weight ratio between GE11-PLGA and PEG-PLGA, obtaining smart nanoplatforms with proper size for parenteral administration (143.9±5.0nm). In vitro cellular uptake in EGFR-overexpressing cell line (A549) demonstrated an active internalization of GE11-functionalized nanoparticles. GE11-PLGA/PEG-PLGA blend nanoparticles were loaded with Myoglobin, a model hydrophilic macromolecule, reaching a good loading (2.42% respect to the theoretical 4.00% w/w) and a prolonged release over 60days. GE11-PLGA/PEG-PLGA blend nanoparticles showed good in vitro stability for 30days in physiological saline solution at 4°C and for 24h in pH 7.4 or pH 5.0 buffer at 37°C respectively, giving indications about potential storage and administration conditions. Furthermore ex vivo stability study in human plasma using fluorescence Single Particle Tracking (fSPT) assessed good GE11-PLGA/PEG-PLGA nanoparticles dimensional stability after 1 and 4h. Thanks to the versatility in polymeric composition and relative tunable nanoparticles features in terms of drug incorporation and release, GE11-PLGA/PEG-PLGA blend NPs can be considered highly promising as smart nanoparticulate platforms for the treatment of diseases characterized by EGFR overexpression by parenteral administration .

Challenging the other: exploring the role of opponent gender in digital game competition for female players.

Abstract The present study investigated the effect of opponent gender on the game experience of female players. Concretely, it looked into skill perception and player emotions of women in same gender and cross-gender game competition. We set up a 2×2×2 (male vs. female opponent×low vs. high competitive women×lost vs. won game) experimental design in which women were instructed to play against a proclaimed male and female competitor. Unknowingly, however, participants played against an AI, which was configured to produce a winning and a losing condition for each opponent by manipulating difficulty. Results indicated that opponent gender only had an effect on perceived stress, which was higher with male opponents. Moreover, players evaluated their own gaming skills as lower and the skills of presumed male opponents as higher when they thought they were playing against men. Importantly, our results also showed that the above described pattern for self-perceived skills and perceived opponent skills was modulated by trait competitiveness with a larger effect size for low competitive women. Overall, this study illustrates that gender dynamics affect the play experience of women in cross-gender gaming competition. Implications and suggestions for future research are discussed.