miRNA Delivery by Nanosystems: State of the Art and Perspectives.

MicroRNAs (miRNAs) are short (~21-23 nucleotides), non-coding endogenous RNA molecules that modulate gene expression at the post-transcriptional level via the endogenous RNA interference machinery of the cell. They have emerged as potential biopharmaceuticals candidates for the treatment of various diseases, including cancer, cardiovascular and metabolic diseases. However, in order to advance miRNAs therapeutics into clinical settings, their delivery remains a major challenge. Different types of vectors have been investigated to allow the delivery of miRNA in the diseased tissue. In particular, non-viral delivery systems have shown important advantages such as versatility, low cost, easy fabrication and low immunogenicity. Here, we present a general overview of the main types of non-viral vectors developed for miRNA delivery, with their advantages, limitations and future perspectives.

P-selectin targeting polysaccharide-based nanogels for miRNA delivery.

Nanogels were prepared in aqueous media without the use of any organic solvent via a simple polyelectrolyte complexation method between aminated pullulan and fucoidan followed by covalent crosslinking with genipin. Homogeneously distributed genipin crosslinked nanogels (G-PECs) were obtained with a mean hydrodynamic diameter of ~155 nm and zeta potential of 0.86 ± 4.35 mV. Their capacity to bind to human activated platelets was evaluated in vitro, as well as their cytocompatibility within human endothelial cells after 1 day of incubation up to 1000 µg/mL of G-PECs (94.56 ± 7.82% of viable cells). Additional hemolysis tests support the biocompatible character of the developed nanosystems (hemolysis rate of 2.09 ± 0.06% for 1000 µg/mL of G-PECs). Under acid conditions, the surface charge of G-PECs was tuned to around ~10 mV allowing miRNA incorporation via electrostatic interactions. G-PECs were able to promote miRNA delivery inside cells, as demonstrated by fluorescence microscopy images of labelled miRNA. With further studies to demonstrate the biological activity of delivered miRNA, these nanogels could be an interesting platform for miRNA-based therapeutics in atherothrombotic-related diseases thanks to the possibility to target over-expressed P-selectin.

Gd(DOTA)-grafted submicronic polysaccharide-based particles functionalized with fucoidan as potential MR contrast agent able to target human activated platelets.

Early detection of thrombotic events remains a big medical challenge. Dextran-based submicronic particles bearing Gd(DOTA) groups and functionalized with fucoidan have been produced via a simple and green water-in-oil emulsification/co-crosslinking process. Their capacity to bind to human activated platelets was evidenced in vitro as well as their cytocompatibility with human endothelial cells. The presence of Gd(DOTA) moieties was confirmed by elemental analysis and total reflection X-ray fluorescence (TRXF) spectrometry. Detailed characterization of particles was performed in terms of size distribution, morphology, and relaxation rates. In particular, longitudinal and transversal proton relaxivities were respectively 1.7 and 5.0 times higher than those of DOTAREM. This study highlights their potential as an MRI diagnostic platform for atherothrombosis.

Synthesis of cationic quaternized pullulan derivatives for miRNA delivery.

Pullulan is a natural polysaccharide of potential interest for biomedical applications due to its non-toxic, non-immunogenic and biodegradable properties. The aim of this work was to synthesize cationic pullulan derivatives able to form complexes with microRNAs (miRNAs) driven by electrostatic interaction (polyplexes). Quaternized ammonium groups were linked to pullulan backbone by adding the reactive glycidyltrimethylammonium chloride (GTMAC). The presence of these cationic groups within the pullulan was confirmed by elemental analysis, Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). The alkylated pullulan was able to interact with miRNA and form stable polyplexes that were characterized regarding size, zeta potential and morphology. The presence of miRNA was confirmed by agarose gel electrophoresis and UV spectrophotometry. In vitro tests on human umbilical vein endothelial cells did not show any cytotoxicity after 1 day of incubation with nanosized polyplexes up to 200 µg/mL. QA-pullulan was able to promote miRNA delivery inside cells as demonstrated by fluorescence microscopy images of labelled miRNA. In conclusion, the formation of polyplexes using cationic derivatives of pullulan with miRNA provided an easy and versatile method for polysaccharide nanoparticle production in aqueous media and could be a new promising platform for gene delivery.

Core-Shell Polymer-Based Nanoparticles Deliver miR-155-5p to Endothelial Cells.

Heart failure occurs in over 30% of the worldwide population and most commonly originates from cardiovascular diseases such as myocardial infarction. microRNAs (miRNAs) target and silence specific mRNAs, thereby regulating gene expression. Because the endogenous miR-155-5p has been ascribed to vasculoprotection, loading it onto positively charged, core-shell poly(isobutylcyanoacrylate) (PIBCA)-polysaccharide nanoparticles (NPs) was attempted. NPs showed a decrease (p < 0.0001) in surface electrical charge (ζ potential), with negligible changes in size or shape when loaded with the anionic miR-155-5p. Presence of miR-155-5p in loaded NPs was further quantified. Cytocompatibility up to 100 µg/mL of NPs for 2 days with human coronary artery endothelial cells (hCAECs) was documented. NPs were able to enter hCAECs and were localized in the endoplasmic reticulum (ER). Expression of miR-155-5p was increased within the cells by 75-fold after 4 hours of incubation (p < 0.05) and was still noticeable at day 2. Differences between loaded NP-cultured cells and free miRNA, at days 1 (p < 0.05) and 2 (p < 0.001) suggest the ability of prolonged load release in physiological conditions. Expression of miR-155-5p downstream target BACH1 was decreased in the cells by 4-fold after 1 day of incubation (p < 0.05). This study is a first proof of concept that miR-155-5p can be loaded onto NPs and remain intact and biologically active in endothelial cells (ECs). These nanosystems could potentially increase an endogenous cytoprotective response and decrease damage within infarcted hearts.

Novel platensimycin derivatives with herbicidal activity.

BACKGROUND: Faced with the need to develop herbicides with different modes of action on account of weed resistance to existing herbicides, the sesquiterpene lactones can be the starting point in the search for new bioactive compounds. Lumisantonin and five novel amides have been evaluated against two monocotyledons and three dicotyledons. RESULTS: An efficient and versatile synthesis of lumisantonin and the five novel amides has been accomplished from readily available α-santonin. These compounds were subjected to evaluation for their biological activity against Sorghum bicolor (sorghum), Allium cepa (onion), Cucumis sativus (cucumber), Solanum lycopersicum (tomato) and Bidens pilosa (beggartick). Lumisantonin has inhibited the development of the aerial parts of sorghum and onion by 76 and 67% at 1000 µM respectively. One of the novel amides has prevented the growth of shoots and radicles of sorghum by 80 and 71% at 1000 µM respectively. CONCLUSION: All of the tested compounds have been found to exhibit promising seed germination inhibition. We can conclude that lumisantonin was on average the most lethal against all plant species evaluated; however, two of the novel amides have exhibited inhibition selectivity against monocotyledons when compared with dicotyledons. © 2015 Society of Chemical Industry.

Quantification of dapaconazole in human plasma using high-performance liquid chromatography coupled to tandem mass spectrometry: application to a phase I study.

A simple, selective and sensitive method based on high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) has been developed for the determination of dapaconazole in human plasma using tioconazole as internal standard. The drugs were extracted from plasma by liquid-liquid extraction with ether/hexane (80/20, v/v). The chromatography separation was performed on a Genesis(®) C18 reversed phase analytical column 4µm (100×2.1mm i.d.) with a mobile phase of methanol/acetonitrile/water (80/10/10, v/v/v)+ammonium acetate (0.5mM). Dapaconazole was quantified using a mass spectrometer with an electrospray source in the ESI positive mode (ES+) configured for multiple reaction monitoring (MRM) to monitor the transitions 415.1>159.2 and 387.0>131.0 for dapaconazole and tioconazole, respectively. The method had a chromatography run time of 3.8min and a linear calibration curve over the range 0.2-100ng/mL (r=0.9998). The lower limit of quantification (LLOQ) was 0.2ng/mL. The precision and accuracy values of the assay were within ±10%. The stability tests indicate no significant degradation under the conditions of the experiment. This method was used for a phase I study of topical administration of dapaconazole tosylate in healthy human male volunteers.