Physicochemical and biological characterization of chitosan-microRNA nanocomplexes for gene delivery to MCF-7 breast cancer cells

Cancer gene therapy requires the design of non-viral vectors that carry genetic material and selectively deliver it with minimal toxicity. Non-viral vectors based on cationic natural polymers can form electrostatic complexes with negatively-charged polynucleotides such as microRNAs (miRNAs). Here we investigated the physicochemical/biophysical properties of chitosan–hsa-miRNA-145 (CS–miRNA) nanocomplexes and the biological responses of MCF-7 breast cancer cells cultured in vitro. Self-assembled CS–miRNA nanocomplexes were produced with a range of (+/−) charge ratios (from 0.6 to 8) using chitosans with various degrees of acetylation and molecular weight. The Z-average particle diameter of the complexes was <200 nm. The surface charge increased with increasing amount of chitosan. We observed that chitosan induces the base-stacking of miRNA in a concentration dependent manner. Surface plasmon resonance spectroscopy shows that complexes formed by low degree of acetylation chitosans are highly stable, regardless of the molecular weight. We found no evidence that these complexes were cytotoxic towards MCF-7 cells. Furthermore, CS–miRNA nanocomplexes with degree of acetylation 12% and 29% were biologically active, showing successful downregulation of target mRNA expression in MCF-7 cells. Our data, therefore, shows that CS–miRNA complexes offer a promising non-viral platform for breast cancer gene therapy

RNA delivery by extracellular vesicles in mammalian cells and its applications.

The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications

Targeted chitosan-based delivery systems of siRNA

The mechanism of RNA silencing has first been evidenced in plants. Then, studies on the nematode Caenorhabditis elegans identified a similar phenomenon, called RNA interference (RNAi). RNAi was described as a naturally occurring mechanism that induced sequence-specific post-transcriptional gene silencing and that was mediated by long double stranded RNAs. Few years later, the administration of exogenous small interfering RNA (siRNA) of 21-22 nucleotides in mammalian cells successfully induced the silencing of endogenous and heterologous genes. This discovery has created new perspectives for the development of gene medicines, especially for the treatment of diseases that are characterized by an overexpression of specific proteins. The RNAi technology promised the rapid development of highly specific therapeutics that could theoretically silence any gene, thus overcoming the issue of undruggable targets. However, after the initial enthusiasm, it clearly appeared that the delivery of small RNAs molecules required precisely engineered nanosystems. Although recent progresses have been made in the understanding of the siRNA delivery modalities, as well as in the design of siRNA delivery systems, the translational development of RNAi-based drugs remains challenging and researchers are still looking for powerful delivery approaches. This work investigated the biopolymer chitosan as the basis material for the elaboration of a siRNA delivery system for tumor targeting. Chitosan exhibits attractive properties, such as its biocompatibility and biodegradability, its cationic nature and its chemical versatility that allows the grafting of targeting ligand. These characteristics make chitosan an ideal basis for nanoparticles (NPs) construction. The research was conducted in three steps. First, the formulation parameters for the design of optimal siRNA NPs using chitosan were established. The principle requirements for ideal NPs were suitable physico-chemical characteristics, especially size and polydispersity, high gene silencing activity without cytotoxicity and stability in plasma. This first part provided the basis for the further design of ligand-grafted NPs. The second part focused on the in vitro evaluation of the targeted nanoparticles and, more precisely, on the investigation of their intracellular delivery mechanisms using a new method of stem-loop RT-qPCR. This phase of the project provided a proof of principle of the cancer-cell targeting ability of our delivery system. Finally, different ligand structures, grafting strategies (clip versus chain-end coupling) and linker properties (hydrophilic versus lipophilic) were investigated to determine whether these parameters influence NP efficacy. Moreover, with the prospect of future in vivo testing, the NPs were evaluated in vitro for their ability to silence the therapeutic targets implicated in cancer metabolism.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Nanoémulsion de fisétine à visée antitumorale

BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Chitosan-based siRNA delivery systems

Recently, chitosan has attracted significant attention in the formulation of small interfering RNA (siRNA). Because of its cationic nature, chitosan can easily complex siRNA, thus readily forming nanoparticles. Moreover, chitosan is biocompatible and biodegradable, which make it a good candidate for siRNA delivery in vivo. However, chitosan requires further development to achieve high efficiency. This review will describe the major barriers that impair the efficiency of the chitosan-based siRNA delivery systems, including the stability of the delivery system in biological fluids and endosomal escape. Several solutions to counteract these barriers have been developed and will be discussed. The parameters to consider for designing powerful delivery systems will be described, particularly the possibilities for grafting targeting ligands. Finally, optimized systems that allow in vivo therapeutic applications for both local and systemic delivery will be reviewed. This review will present recent improvements in chitosan-based siRNA delivery systems that overcome many of these system's previous pitfalls and pave the way to a new generation of siRNA delivery systems

Chitosan Nanoparticles for SiRNA Delivery In Vitro.

RNA interference, the process in which small interfering RNAs (SiRNAs) silence a specific gene and thus inhibit the associated protein, has opened new doors for the treatment of a wide range of diseases. However, efficient delivery of SiRNAs remains a challenge, especially due to their instability in biological environments and their inability to cross cell membranes. To protect and deliver SiRNAs to mammalian cells, a variety of polymeric nanocarriers have been developed. Among them, the polysaccharide chitosan has generated great interests. This derivative of natural chitin is biodegradable and biocompatible, and can complex SiRNAs into nanoparticles on account of its positive charges. However, chitosan presents some limitations that need to be taken into account when designing chitosan/SiRNA nanoparticles. Here, we describe a method to prepare SiRNA/chitosan nanoparticles with high gene silencing efficiency and low cytotoxicity by using the ionic gelation technique

Nanoparticle-based drug delivery systems: a commercial and regulatory outlook as the field matures.

Nanomedicine has emerged as a major field of academic research with direct impact on human health. While a first generation of products has been successfully commercialized and has significantly contributed to enhance patient's life, recent advances in material design and the emergence of new therapeutics are contributing to the development of more sophisticated systems. As the field matures, it is important to comprehend the challenges related to nanoparticle commercial development for a more efficient and predictable path to the clinic. Areas covered: The review provides an overview of nanoparticle-based delivery systems currently on the market and in clinical trials, and discuss the principal challenges for their commercial development, both from a manufacturing and regulatory perspective, to help gain understanding of the translational path for these systems. Expert Opinion: Clinical translation of nanoparticle-based delivery systems remains challenging on account of their 3D nanostructure and requires robust nano-manufacturing process along with adequate analytical tools and methodologies. By identifying early enough in the development the product critical attributes and understanding their impact on the therapeutic performance, the developers of nanopharmaceuticals will be better equipped to develop efficient product pipelines. Second-generation products are expected to broaden nanopharmaceutical global market in the upcoming years