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

Aerosolization of nanotherapeutics as a newly emerging treatment regimen for peritoneal carcinomatosis

Recent advances in locoregional chemotherapy have opened the door to new approaches for the clinical management of peritoneal carcinomatosis (PC) by facilitating the delivery of anti-neoplastic agents directly to the tumor site, while mitigating adverse effects typically associated with systemic administration. In particular, an innovative intra-abdominal chemotherapeutic approach, known as Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC), was recently introduced to the intraperitoneal (IP) therapy regimens as a palliative therapeutic option in patients with PC, presumably providing a better drug distribution pattern together with deeper drug penetration into tumor nodules within the peritoneal space. Furthermore, the progress of nanotechnology in the past few decades has prompted the application of different nanomaterials in IP cancer therapy, offering new possibilities in this field ranging from an extended retention time to sustained drug release in the peritoneal cavity. This review highlights the progress, challenges, and opportunities in utilizing cancer nanotherapeutics for locoregional drug delivery, with a special emphasis on the aerosolization approach for intraperitoneal therapies

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

Intraperitoneal chemotherapy for peritoneal metastases : an expert opinion

Introduction: The rationale for intraperitoneal (IP) drug delivery for patients with peritoneal metastases (PM) is based on the pharmacokinetic advantage resulting from the peritoneal-plasma barrier, and on the potential to adequately treat small, poorly vascularized PM. Despite a history of more than three decades, many aspects of IP drug delivery remain poorly studied. Areas covered: We outline the anatomy and physiology of the peritoneal cavity, including the pharmacokinetics of IP drug delivery. We discuss transport mechanisms governing tissue penetration of IP chemotherapy, and how these are affected by the biomechanical properties of the tumor stroma. We provide an overview of the current clinical evidence on IP chemotherapy in ovarian, colorectal, and gastric cancer. We discuss the current limitations of IP drug delivery and propose several potential areas of progress. Expert opinion: The potential of IP drug delivery is hampered by off-label use of drugs developed for systemic therapy. The efficacy of IP chemotherapy for PM depends on cancer type, disease extent, and mode of drug delivery. Results from ongoing randomized trials will allow to better delineate the potential of IP chemotherapy. Promising approaches include IP aerosol therapy, prolonged delivery platforms such as gels or biomaterials, and the use of nanomedicine

Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry

P pili are multisubunit fibers essential for the attachment of uropathogenic Escherichia coli to the kidney. These fibers are formed by the noncovalent assembly of six different homologous subunit types in an array that is strictly defined in terms of both the number and order of each subunit type. Assembly occurs through a mechanism termed “donor-strand exchange (DSE)” in which an N-terminal extension (Nte) of one subunit donates a β-strand to an adjacent subunit, completing its Ig fold. Despite structural determination of the different subunits, the mechanism determining specificity of subunit ordering in pilus assembly remained unclear. Here, we have used noncovalent mass spectrometry to monitor DSE between all 30 possible pairs of P pilus subunits and their Ntes. We demonstrate a striking correlation between the natural order of subunits in pili and their ability to undergo DSE in vitro. The results reveal insights into the molecular mechanism by which subunit ordering during the assembly of this complex is achieved

Nuclear inclusion of nontargeted and chromatin-targeted polystyrene beads and plasmid DNA containing nanoparticles

The nuclear membrane is one of the major cellular barriers in the delivery of plasmid DNA (pDNA). Cell division has a positive influence on the expression efficiency since, at the end of mitosis, pDNA or pDNA containing complexes near the chromatin are probably included by a random process in the nuclei of the daughter cells. However, very little is known about the nuclear inclusion of nanoparticles during cell division. Using the Xenopus nuclear envelope reassembly (XNER) assay, we found that the nuclear enclosure of nanoparticles was dependent on size (with 100 and 200 nm particles being better included than the 500 nm ones) and charge (with positively charged particles being better included than negatively charged cr polyethyleneglycolated (PEGylated) ones) of the beads. Also, coupling chromatin-targeting peptides to the polystyrene beads or pDNA complexes improved their inclusion by 2- to 3-fold. Upon microinjection in living HeLa cells, however, nanoparticles were never observed in the nuclei of cells postdivision but accumulated in a specific perinuclear region, which was identified as the lysosomal compartment. This indicates that nanoparticles can end up in the lysosomes even when they were not delivered through endocytosis. To elucidate if the chromatin binding peptides also have potential in living cells, this additional barrier first has to be tackled, since it prevents free particles from being present near the chromatin at the moment of cell division

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