Fluorescent labeling of plasmid DNA and mRNA : gains and losses of current labeling strategies

Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this Review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular mRNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs

The influence of movement on the localization precision of sub-resolution particles in fluorescence microscopy

Obtaining sub-resolution particle positions in fluorescence microscopy images is essential for single particle tracking and high-resolution localization microscopy. While the localization precision of stationary single molecules or particles is well understood, the influence of particle motion during image acquisition has been largely neglected. Here, we address this issue and provide a theoretical description on how particle motion influences the centroid localization precision, both in case of 2-D and 3-D diffusion. In addition, a novel method is proposed, based on dual-channel imaging, for the experimental determination of the localization precision of moving particles. For typical single particle tracking experiments, we show that the localization precision is approximately two-fold worse than expected from the stationary theory. Strikingly, we find that the most popular localization method, based on the fitting of a Gaussian distribution, breaks down for lateral diffusion. Instead, the centroid localization method is found to perform well under all conditions. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Exploring the HYDRAtion method for loading siRNA on liposomes : the interplay between stability and biological activity in human undiluted ascites fluid

Delivery of small interfering RNA (siRNA) is recently gaining tremendous attention for the treatment of ovarian cancer. The present study investigated the potential of different liposomal formulations composed of (2,3-dioleoyloxy-propyl)trimethylammonium (DOTAP) and 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE) encapsulating siRNA (hydration method) for their ability to knockdown luciferase (Luc) activity in human ovarian cancer SKOV-3 cells. Fluorescence single particle tracking (fSPT) and fluorescence correlation spectroscopy (FCS) in human-undiluted ascites fluid obtained from a peritoneal carcinomatosis patient revealed that cationic hydra-lipoplexes (HYDRA-LPXs) and HYDRA-LPXs decorated with stable DSPE-PEG (DSPE HYDRA-LPXs) showed high stability during at least 24 h. HYDRA-LPXs decorated with sheddable C8 and C16 PEG-Ceramides (Cer HYDRA-LPXs) resulted in rapid and premature release of siRNA already in the first hours. Despite their role in preventing aggregation in vivo, liposomes decorated with stable PEG residues resulted in a poor transfection compared to the ones decorated with sheddable PEG residues in reduced serum conditions. Yet, the transfection efficiency of both Cer HYDRA-LPXs significantly decreased following 1 h of incubation in ascites fluid due to a drastic drop in the cellular uptake, while DSPE HYDRA-LPXs are still taken up by cells, but too stable to induce efficient gene silencing

The influence of natural pulmonary surfactant on the efficacy of siRNA-loaded dextran nanogels

Aim: Topical administration of siRNA nanocarriers is a promising approach in the treatment of pulmonary disorders. Pulmonary surfactant, covering the entire alveolar surface of mammalian lungs, will be one of the first interfaces that siRNA nanocarriers encounter upon inhalation therapy. Therefore, it is of outstanding importance to evaluate the impact of pulmonary surfactant on the performance of siRNA nanocarriers. Materials & methods: The effect of natural lung-derived surfactants on the siRNA delivery capacity of dextran nanogels (DEX-NGs) was evaluated in vitro using flow cytometry and confocal microscopy. Results: Although the interaction with pulmonary surfactant decreases the cellular internalization of siRNA-loaded DEX-NGs significantly, the gene silencing potential of siRNA-loaded DEX-NGs was maintained. On the other hand, cationic lipid-based siRNA nanocarriers (Lipofectamine (TM) RNAiMAX) were incompatible with pulmonary surfactants. Conclusion: Our data suggest that pulmonary surfactant can enhance the intracellular siRNA delivery by DEX-NGs, thereby possibly providing new therapeutic opportunities

Proactive resource allocation heuristics for robust project scheduling.

The well-known deterministic resource-constrained project scheduling problem (RCPSP) involves the determination of a predictive schedule (baseline schedule or pre-schedule) of the project activities that satisfies the finish-start precedence relations and the renewable resource constraints under the objective of minimizing the project duration. This pre-schedule serves as a baseline for the execution of the project. During execution, however, the project can be subject to several types of disruptions that may disturb the baseline schedule. Management must then rely on a reactive scheduling procedure for revising or reoptimizing the pre-schedule. The objective of our research is to develop procedures for allocating resources to the activities of a given baseline schedule in order to maximize its stability. We propose two integer programming based heuristics and report on computational results obtained on a set of benchmark problems.

Disregarded effect of biological fluids in siRNA delivery : human ascites fluid severely restricts cellular uptake of nanoparticles

Small interfering RNA (siRNA) offers a great potential for the treatment of various diseases and disorders. Nevertheless, inefficient in vivo siRNA delivery hampers its translation into the clinic. While numerous successful in vitro siRNA delivery stories exist in reduced-protein conditions, most studies so far overlook the influence of the biological fluids present in the in vivo environment. In this study, we compared the transfection efficiency of liposomal formulations in Opti-MEM (low protein content, routinely used for in vitro screening) and human undiluted ascites fluid obtained from a peritoneal carcinomatosis patient (high protein content, representing the in vivo situation). In Opti-MEM, all formulations are biologically active. In ascites fluid, however, the biological activity of all lipoplexes is lost except for lipofectamine RNAiMAX. The drop in transfection efficiency was not correlated to the physicochemical properties of the nanoparticles, such as premature siRNA release and aggregation of the nanoparticles in the human ascites fluid. Remarkably, however, all of the formulations except for lipofectamine RNAiMAX lost their ability to be taken up by cells following incubation in ascites fluid. To take into account the possible effects of a protein corona formed around the nanoparticles, we recommend always using undiluted biological fluids for the in vitro optimization of nanosized siRNA formulations next to conventional screening in low-protein content media. This should tighten the gap between in vitro and in vivo performance of nanoparticles and ensure the optimal selection of nanoparticles for further in vivo studies

Spatiotemporal visualization of subcellular dynamics of carbon nanotubes

To date, there is no consensus on the relationship between the physicochemical characteristics of carbon nanotubes (CNTs) and their biological behavior; however, there is growing evidence that the versatile characteristics make their biological fate largely unpredictable and remain an issue of limited knowledge. Here we introduce an experimental methodology for tracking and visualization of post-uptake behavior and the intracellular fate of CNTs based on the spatial distribution of diffusion values throughout the plant cell. By using raster scan image correlation spectroscopy (RICS), we were able to generate highly quantitative spatial maps of CNTs diffusion in different cell compartments. The spatial map of diffusion values revealed that the uptake of CNTs is associated with important subcellular events such as carrier-mediated vacuolar transport and autophagy. These results show that RICS is a useful methodology to elucidate the intracellular behavior mechanisms of carbon nanotubes and potentially other fluorescently labeled nanoparticles, which is of relevance for the important issues related to the environmental impact and health hazards

Laser-assisted photoporation : fundamentals, technological advances and applications

Laser-assisted photoporation is a promising technique that is receiving increasing attention for the delivery of membrane impermeable nanoscopic substances into living cells. Photoporation is based on the generation of localized transient pores in the cell membrane using continuous or pulsed laser light. Increased membrane permeability can be achieved directly by focused laser light or in combination with sensitizing nanoparticles for higher throughput. Here, we provide a detailed account on the history and current state-of-the-art of photoporation as a physical nanomaterial delivery technique. We first introduce with a detailed explanation of the mechanisms responsible for cell membrane pore formation, following an overview of experimental procedures for realizing direct laser photoporation. Next, we review the second and most recent method of photoporation that combines laser light with sensitizing NPs. The different mechanisms of pore formation are discussed and an overview is given of the various types of sensitizing nanomaterials. Typical experimental setups to achieve nanoparticle-mediated photoporation are discussed as well. Finally, we discuss the biological and therapeutic applications enabled by photoporation and give our current view on this expanding research field and the challenges and opportunities that remain for the near future