Targeted nanoparticles towards increased L cell stimulation as a strategy to improve oral peptide delivery in incretin-based diabetes treatment

The delivery of therapeutic peptides via the oral route remains one of biggest challenges in the pharmaceutical industry. Recently, we have described an alternative improved drug delivery system for peptide delivery via the oral route, consisting of a lipidic nanocapsule. Despite the striking effects observed, it is still essential to develop strategies to strengthen the nanocarriers' glucagon-like peptide-1 (GLP-1) secretory effect of the nanocarrier and/or prolong its antidiabetic effect in vivo to facilitate its translation into the clinic. For this purpose, we developed and compared different fatty acid-targeted lipid and polymeric nanoparticles and evaluated the L cell stimulation induced by the nanocarriers in murine L cells in vitro and in normal healthy mice in vivo. We further examined the antidiabetic effect in vivo in an obese/diabetic mouse model induced by high-fat diet feeding and examined the effect of the oral administration frequency. Among the tested nanocarriers, only lipid-based nanocarriers that were surface-modified with DSPE-PEG(2000) on the surface were able to significantly strengthen the biological effect of the nanocarriers. They increased endogenous GLP-1 levels up to 8-fold in vivo in normo-glycemic mice. Moreover, they effectively prolonged the in vivo antidiabetic effect by normalizing the plasma glucose levels in obese/diabetic mice following long-term treatment (one month). Ultimately, the targeted nanocarriers were as effective when the administration frequency was reduced from once daily to once every other day

Layer by layer assembled chitosan-coated gold nanoparticles for enhanced siRNA delivery and silencing

Delivery of small interfering RNA (siRNA) provides one of the most powerful strategies for downregulation of therapeutic targets. Despite the widely explored capabilities of this strategy, intracellular delivery is hindered by a lack of carriers that have high stability, low toxicity and high transfection efficiency. Here we propose a layer by layer (LBL) self-assembly method to fabricate chitosan-coated gold nanoparticles (CS-AuNPs) as a more stable and efficient siRNA delivery system. Direct reduction of HAuCl4 in the presence of chitosan led to the formation of positively charged CS-AuNPs, which were subsequently modified with a layer of siRNA cargo molecules and a final chitosan layer to protect the siRNA and to have a net positive charge for good interaction with cells. Cytotoxicity, uptake, and downregulation of enhanced Green Fluorescent Protein (eGFP) in H1299-eGFP lung epithelial cells indicated that LBL-CS-AuNPs provided excellent protection of siRNA against enzymatic degradation, ensured good uptake in cells by endocytosis, facilitated endosomal escape of siRNA, and improved the overall silencing effect in comparison with commercial transfection reagents Lipofectamine and jetPEI(R). Therefore, this work shows that LBL assembled CS-AuNPs are promising nanocarriers for enhanced intracellular siRNA delivery and silencing

The role of small proteins in Burkholderia cenocepacia J2315 biofilm formation, persistence and intracellular growth

Burkholderia cenocepacia infections are difficult to treat due to resistance, biofilm formation and persistence. B. cenocepacia strain J2315 has a large multi-replicon genome (8.06 Mb) and the function of a large fraction of (conserved) hypothetical genes remains elusive. The goal of the present study is to elucidate the role of small proteins in B. cenocepacia, focusing on genes smaller than 300 base pairs of which the function is unknown. Almost 10% (572) of the B. cenocepacia J2315 genes are smaller than 300 base pairs and more than half of these are annotated as coding for hypothetical proteins. For 234 of them no similarity could be found with non-hypothetical genes in other bacteria using BLAST. Using available RNA sequencing data obtained from biofilms, a list of 27 highly expressed B. cenocepacia J2315 genes coding for small proteins was compiled. For nine of them expression in biofilms was also confirmed using LC-MS based proteomics and/or expression was confirmed using eGFP translational fusions. Overexpression of two of these genes negatively impacted growth, whereas for four others overexpression led to an increase in biofilm biomass. Overexpression did not have an influence on the MIC for tobramycin, ciprofloxacin or meropenem but for five small protein encoding genes, overexpression had an effect on the number of persister cells in biofilms. While there were no significant differences in adherence to and invasion of A549 epithelial cells between the overexpression mutants and the WT, significant differences were observed in intracellular growth/survival. Finally, the small protein BCAM0271 was identified as an antitoxin belonging to a toxin-antitoxin module. The toxin was found to encode a tRNA acetylase that inhibits translation. In conclusion, our results confirm that small proteins are present in the genome of B. cenocepacia J2315 and indicate that they are involved in various biological processes, including biofilm formation, persistence and intracellular growth.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Enhanced siRNA delivery and selective apoptosis induction in H1299 cancer cells by layer-by-layer-assembled Se nanocomplexes : toward more efficient cancer therapy

Nanotechnology has made an important contribution to oncology in recent years, especially for drug delivery. While many different nano-delivery systems have been suggested for cancer therapy, selenium nanoparticles (SeNPs) are particularly promising anticancer drug carriers as their core material offers interesting synergistic effects to cancer cells. Se compounds can exert cytotoxic effects by acting as pro-oxidants that alter cellular redox homeostasis, eventually leading to apoptosis induction in many kinds of cancer cells. Herein, we report on the design and synthesis of novel layer-by-layer Se-based nanocomplexes (LBL-Se-NCs) as carriers of small interfering RNA (siRNA) for combined gene silencing and apoptosis induction in cancer cells. The LBL-Se-NCs were prepared using a straightforward electrostatic assembly of siRNA and chitosan (CS) on the solid core of the SeNP. In this study, we started by investigating the colloidal stability and protection of the complexed siRNA. The results show that CS not only functioned as an anchoring layer for siRNA, but also provided colloidal stability for at least 20 days in different media when CS was applied as a third layer. The release study revealed that siRNA remained better associated with LBL-Se-NCs, with only a release of 35% after 7 days, as compared to CS-NCs with a siRNA release of 100% after 48 h, making the LBL nanocarrier an excellent candidate as an off-the-shelf formulation. When applied to H1299 cells, it was found that they can selectively induce around 32% apoptosis, while significantly less apoptosis (5.6%) was induced in NIH/3T3 normal cells. At the same time, they were capable of efficiently inducing siRNA downregulation (35%) without loss of activity 7 days post-synthesis. We conclude that LBL-Se-NCs are promising siRNA carriers with enhanced stability and with a dual mode of action against cancer cells

Super-resolution fluorescence microscopy based techniques for the study of signal transduction

To transmit a message from one to another, correct communication is crucial. To obtain correct communication, it is important that both transmitter and receiver of the signal are present at the correct time at the correct location. In a cell this communication is taken care of by signal transduction pathways. They consist of a highly branched network of proteins and messenger molecules, integrating signals to a final work plan guiding the cell in its functions mediating both short- and long-term responses. Therefore, proper distribution in time and space of the components of these networks is crucial for cell behaviour and fate. Single molecule fluorescence microscopy has been proven an excellent tool to study distributions and behaviour of single molecules in time and space providing detection of heterogeneities otherwise lost in ensemble averaging. The objectives of this dissertation were to expand and use fluorescence microscopy tools to study signal transduction in mammalian cultured cells at a high spatial resolution as well as on a single molecule level with as a case study, the EGFR/ MAPK signal transduction pathway. The results are divided in two parts. The first part, 'Fluorescent probes for microscopy', explores the utility of single emission band optical highlighting with LSSmOrange (chapter 5). This study shows that LSSmOrange can be photoconverted in living cells with 'blue' light irradiation. Since minimal binding to endogenous proteins was observed, LSSmOrange is suitable for optical highlighting in cellulo. In addition, highlighting experiments were easily expanded to multicolour imaging due to an invariable emission band. Furthermore, confined photoconversion of LSSmOrange was achieved in three dimensions with a femtosecond laser pulse via a two-photon process. The second part, 'Single molecule fluorescence microscopy applied to signal transduction', consists of chapters 6-8. These chapters deals with single molecule methods to study signal transduction. Protein-protein interactions (PPIs) form the basis of cellular processes regulating cell fate and can be extremely transient. In chapter 6, an approach to detect these short-lived transient interactions on the plasma membrane in living cells was developed. This approach is based on selective detection of interacting single molecules. This was possible due to the difference in mobility between proteins on the membrane and in the cytosol, and therefore decrease in motion blur of the fluorescence signal. The proof of concept was performed with a model system based on modular protein domains interacting with short linear peptide motifs. Interacting molecules are detected and localized. By accumulation of all localizations, an interaction map at the nanoscale was drawn. This approach was then used to image the interaction of several cytosolic proteins involved in the EGF signalling pathway with the plasma membrane. Interaction maps of Grb2, PLC-γ and c-Raf show that these molecules interact with different compartments at the plasma membrane, illustrating the role of membrane heterogeneity in the spatial regulation of cell signalling. In chapter 7, we use this method to investigate the role of receptor distribution in clathrin coated pits on the membrane and on vesicles in the cytosol. Clathrin mediated endocytosis (CME) serves to downregulate membrane receptors but several studies show involvement of this compartmentalization in signalling. Downstream molecules interact with the receptor during signal transduction. To observe the role of EGFR enriched pits and vesicles due to CME, we recorded c-Raf interactions throughout the cell together with the EGFR or clathrin distribution. An analysis method to correlate the receptor distribution with the c-Raf interactions was developed. No increased number of interacting c-Raf molecules was observed located on clathrin coated pits (CCPs) or cytosolic vesicles (CCVs) suggesting that neither on the basal plasma membrane or in the cytosol, EGFR accumulation in CCPs and CCVs played a role as a platform where c-Raf molecules are efficiently binding and activated. In the last chapter (chapter 8), molecular mobility is linked with the cellular response. A cell is a complex entity and response to a trigger can be inhomogeneous between cells. This response is regulated on the molecular level. Proteins such as receptors on the plasma membrane move around and this movement is linked to molecular interactions and the protein environment. To analyze the molecular dynamics in relation to the response, a system was developed to perform functional imaging during aquisition for SPT analysis and applied to the EGFR in its heterogeneous Ca2+ response upon a trigger with submaximal concentration of EGF. Different diffusion properties of the EGFR were revealed in responding and non-responding cells which is thought to reflect the shift in monomer–dimer equilibrium of the EGFR.status: publishe

A Bimolecular Fluorescence Complementation Tool for Identification of Protein-Protein Interactions in Candida albicans

Investigation of protein-protein interactions (PPI) in Candida albicans is essential for understanding the regulation of the signal transduction network that triggers its pathogenic lifestyle. Unique features of C. albicans, such as the alternative codon usage and incomplete meiosis, have enforced the optimization of standard genetic methods as well as development of novel approaches. Since the existing methods for detection of PPI are limited for direct visualization of the interacting complex in vivo, we have established a bimolecular fluorescence complementation (BiFC) in C. albicans, a powerful technique for studying PPI. We have developed an optimized set of plasmids that allows for N- and C-terminal tagging of proteins with split yeast-enhanced monomeric Venus fragments, so that all eight combinations of fusion orientations can be analyzed. With the use of our BiFC assay we demonstrate three interaction complexes in vivo, which were also confirmed by two-hybrid analysis. Our Candida optimized BiFC assay represents a useful molecular tool for PPI studies and shows great promise in expanding the knowledge on molecular mechanisms of protein functions.status: publishe

A Bimolecular Fluorescence Complementation Tool for Identification of Protein-Protein Interactions in Candida albicans

Investigation of protein-protein interactions (PPI) in Candida albicans is essential for understanding the regulation of the signal transduction network that triggers its pathogenic lifestyle. Unique features of C. albicans, such as its alternative codon usage and incomplete meiosis, have enforced the optimization of standard genetic methods as well as development of novel approaches. Since the existing methods for detection of PPI are limited for direct visualization of the interacting complex in vivo, we have established a bimolecular fluorescence complementation (BiFC) assay in C. albicans, a powerful technique for studying PPI. We have developed an optimized set of plasmids that allows for N- and C-terminal tagging of proteins with split yeast-enhanced monomeric Venus fragments, so that all eight combinations of fusion orientations can be analyzed. With the use of our BiFC assay we demonstrate three interaction complexes in vivo, which were also confirmed by two-hybrid analysis. Our Candida-optimized BiFC assay represents a useful molecular tool for PPI studies and shows great promise in expanding our knowledge of molecular mechanisms of protein functions

PEGylation of recombinant human deoxyribonuclease I decreases its transport across lung epithelial cells and uptake by macrophages.

Conjugation to high molecular weight (MW) polyethylene glycol (PEG) was previously shown to largely prolong the lung residence time of recombinant human deoxyribonuclease I (rhDNase) and improve its therapeutic efficacy following pulmonary delivery in mice. In this paper, we investigated the mechanisms promoting the extended lung retention of PEG-rhDNase conjugates using cell culture models and lung biological media. Uptake by alveolar macrophages was also assessed in vivo. Transport experiments showed that PEGylation reduced the uptake and transport of rhDNase across monolayers of Calu-3 cells cultured at an air-liquid interface. PEGylation also decreased the uptake of rhDNase by macrophages in vitro whatever the PEG size as well as in vivo 4 h following intratracheal instillation in mice. However, the reverse was observed in vivo at 24 h. The uptake of rhDNase by macrophages was dependent on energy, time, and concentration and occurred at rates indicative of adsorptive endocytosis. The diffusion of PEGylated rhDNase in porcine tracheal mucus and cystic fibrosis sputa was slower compared with that of rhDNase. Nevertheless, no significant binding of PEGylated rhDNase to both media was observed. In conclusion, decreased transport across lung epithelial cells and uptake by macrophages appear to contribute to the longer retention of PEGylated rhDNase in the lungs

Orthogonal Probing of Single-Molecule Heterogeneity by Correlative Fluorescence and Force Microscopy

Correlative imaging by fluorescence and force microscopy is an emerging technology to acquire orthogonal information at the nanoscale. Whereas atomic force microscopy excels at resolving the envelope structure of nanoscale specimens, fluorescence microscopy can detect specific molecular labels, which enables the unambiguous recognition of molecules in a complex assembly. Whereas correlative imaging at the micrometer scale has been established, it remains challenging to push the technology to the single-molecule level. Here, we used an integrated setup to systematically evaluate the factors that influence the quality of correlative fluorescence and force microscopy. Optimized data processing to ensure accurate drift correction and high localization precision results in image registration accuracies of ∼25 nm on organic fluorophores, which represents a 2-fold improvement over the state of the art in correlative fluorescence and force microscopy. Furthermore, we could extend the Atto532 fluorophore bleaching time ∼2-fold, by chemical modification of the supporting mica surface. In turn, this enables probing the composition of macromolecular complexes by stepwise photobleaching with high confidence. We demonstrate the performance of our method by resolving the stoichiometry of molecular subpopulations in a heterogeneous EcoRV-DNA nucleoprotein ensemble.status: publishe